Artificial insemination method for improving fertilization rate of hapalogenys nitens

Abstract

The invention discloses an artificial insemination method for improving the fertilization rate of hapalogenys nitens, and relates to the technical field of aquaculture, and the method comprises the following steps: selecting healthy parent fishes of 3 years old or above according to the female-male ratio of 2: 1, and culturing; an oxytocic is injected by adopting a two-needle method, LRH-A3 is injected in the first needle, LRH-A3 + HCG is injected in the second needle, the injection amount of the male fish is half of that of the female fish, and the injection interval time is 11-13 h; after induced spawning is conducted for 24 h, female fishes with mature ovaries are screened, bellies are pressed for artificial egg squeezing, and fertilized eggs are obtained through dry-method insemination; the fertilized eggs are transferred into a conical hatching barrel, preferably high-quality fertilized eggs floating on the water surface. By regulating and controlling the environment and oxytocin to synchronously carry out gonad development on the parent fishes of the hapalogenys, accurately screening the female fishes with mature ovaries by utilizing morphological characteristics and assisting with an artificial insemination and high-quality fertilized egg screening method, the egg laying amount is remarkably increased, the fertilization rate is increased to 70% or above compared with a natural fertilization mode, and the hatching rate reaches 95%; and large-scale stable acquisition of high-quality fertilized eggs can be realized.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, in particular to an artificial insemination method for improving the fertilization rate of bream. Background Art

[0002] The cross-banded bream (Hapalogenys mucronatus) is a marine fish with important economic value. It belongs to the order Perciformes, family Pamadasyidae, and genus Hapalogenys. It is commonly known as the "sixteen pieces". It mainly inhabits rocky reef areas and is a typical reef fish. In the consumer market, this fish species is regarded as a high-end seafood delicacy in the Jiangsu, Zhejiang and Shanghai regions because of its tender meat and unique flavor. Nowadays, the main way to obtain cross-banded bream is wild fishing, but the catch of wild resources cannot meet market demand.

[0003] Since the research on artificial large-scale breeding of the horizontal banded bream was launched in 2017, although a breakthrough was achieved in small-scale seedling cultivation in 2020, its extremely low fertilization rate is still the main problem in the large-scale breeding of the horizontal banded bream. Specifically, this species has problems such as asynchronous development of male and female gonads and low amount of male semen, which makes it difficult for females to pair up and fertilize during batch spawning. The Chinese patent application publication number is CN111296330B, and the authorization publication date is October 8, 2021. The invention discloses a device and method for breeding broodstock and obtaining fertilized eggs of horizontal banded bream. The invention uses 100 female fish and 50 male fish for artificial induction of spawning and natural fertilization. Although the theoretical hatching rate can reach 92%, the natural fertilization rate is extremely low, and many problems are exposed in the actual application process: the output of fertilized eggs of horizontal banded bream is unstable, the daily average fluctuation is large, and even the phenomenon of being unable to harvest fertilized eggs for many consecutive days occurs; and the natural fertilization process can last up to 27 days, and manual monitoring and batch egg collection operations are required during the process; the fertilized eggs are produced in small quantities, and the average daily output of fertilized eggs per female fish is only 160. These problems have seriously affected the stable supply of horizontal banded bream seedlings, causing horizontal banded bream farming to still be highly dependent on wild seedling resources. Summary of the invention

[0004] In order to solve the technical problem caused by the extremely low fertilization rate of the horizontal banded bream for its large-scale breeding, the present invention provides an artificial insemination method for improving the fertilization rate of the horizontal banded bream. The present invention synchronizes the gonadal development of parent fish by combining environmental regulation with injection of oxytocin, accurately screens female fish with mature ovaries by using morphological characteristics, and supplements with artificial insemination and high-quality fertilized egg screening methods to significantly increase the spawning amount of the horizontal banded bream. The fertilization rate is increased to more than 70% compared with the natural fertilization method, and the hatching rate reaches 95%, which can realize the large-scale stable acquisition of high-quality fertilized eggs. The technical scheme adopted is as follows:

[0005] An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus, comprising the following steps:

[0006] (1) Select healthy female and male fish aged 3 or above, with a female-to-male ratio of 1:(1.5 - 2.5), and put them into the broodstock pond for cultivation to obtain the broodstock to be induced to spawn;

[0007] (2) The broodstock to be induced to spawn is injected with the spawning inducer by the two-needle method. The first needle injects LRH-A3, and the second needle injects LRH-A3 + HCG. The injection amount for male fish is half of that for female fish, and the injection interval is 11h - 13h;

[0008] (3) 24 hours after injecting the spawning inducer, select female fish with mature ovarian development, press the abdomen to perform artificial egg extrusion, and obtain fertilized eggs by dry insemination;

[0009] (4) Transfer the fertilized eggs to a conical hatching barrel, and use a 60-mesh silk screen to collect the high-quality fertilized eggs floating on the water surface and place them in the hatching pond for hatching.

[0010] Preferably, in the step (1), the broodstock pond is a circular cement pond with a water depth of 50 - 80 cm and an area of 15 - 20 m 2 , equipped with a temperature control, lighting, oxygenation and inlet and outlet water facility system; the water inlet forms a 45° angle with the pool wall along the same direction, and the drain outlet is located at the center of the pool bottom; the dissolved oxygen in the water body is 5 - 8 mg / L, the salinity is 24 - 28, the pH is 7.5 - 8.2, the water is changed twice a day, the water change amount is 200%, and the sewage is sucked once in the morning and evening each.

[0011] Preferably, in the step (2), the injection dose of LRH-A3 for the first needle of female fish is 0.5 - 1.5 μg / kg; the injection dose of LRH-A3 for the second needle of female fish is 1 - 3 μg / kg, and the injection dose of HCG is 50 - 150 IU / kg.

[0012] Preferably, in the step (2), when injecting the spawning inducer, the head of the broodstock is wrapped with a wet towel soaked in seawater, and thoracic injection is adopted, and the injection position is the base of the pectoral fin of the broodstock.

[0013] Preferably, in the step (2), the water temperature of the broodstock pond is 23°C after injecting the first needle of the spawning inducer, and a sunshade net is covered on the broodstock pond; the water temperature of the broodstock pond is adjusted to 25°C after injecting the second needle, and the sunshade net is still covered; the breeding workshop is kept quiet, and at the same time, the feeding of bait is stopped.

[0014] Preferably, in the step (3), the criteria for selecting female fish with mature ovarian development are that when touching the abdomen of the female fish by hand, it feels soft; the eggs are felt to be separated and flowing in the ovary; the genital pore protrudes after gently pressing the abdomen of the female fish.

[0015] Preferably, in the step (3), the operation cycle for selecting female fish with mature ovarian development is once every 30 - 60 minutes.

[0016] Preferably, in the dry fertilization operation in step (3), a clean towel is used to dry the abdomen of the female fish, and the abdomen is gently squeezed by hand to squeeze the mature eggs into a washbasin that is clean, dry and shielded from strong light. Then, the semen is squeezed onto the fish eggs, and gently stirred evenly with a feather for 30 s. Sand-filtered seawater is added, and stirring continues evenly for 30 s. Then, sand-filtered seawater is slowly added and stirred evenly for 1 min to allow the eggs and sperm to fully combine. It is left to stand for 4-5 min, and then the excess semen is filtered off with a 60-mesh silk screen, and the eggs are washed with seawater 2-3 times.

[0017] More preferably, in the dry fertilization operation in step (3), the volume ratio of eggs, semen and sand-filtered seawater is 1000:(1-2):2500; the semen is collected from 2-3 male fish.

[0018] More preferably, in step (4), the time for collecting high-quality fertilized eggs is 5-7 hours after the fertilized eggs are placed in the hatching barrel. After the high-quality fertilized eggs are placed in the hatching pond for 28-32 hours, newly hatched larvae are obtained.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The male-female ratio of the broodstock population is regulated, and the proportion of male broodstock is increased by screening the broodstock, effectively solving the problem of insufficient semen volume in male individuals; (2) The oxytocin ratio and injection method are optimized, combined with the management of the broodstock growth environment, to synchronize the gonadal development of the broodstock and reduce the damage of exogenous hormones to the broodstock; (3) By giving the morphological standards of the female Hapalogenys mucronatus with mature ovaries, the best egg collection time is grasped, and mature eggs with high fertilization vitality are accurately obtained; (4) The sperm-egg mixing ratio is optimized in the artificial insemination operation to improve the fertilization rate. Description of the Drawings

[0020] Figure 1 The juvenile Hapalogenys mucronatus bred by the method of this application. Detailed Embodiments

[0021] The present invention will be further described below in conjunction with specific embodiments:

[0022] General Embodiment

[0023] An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus, comprising the following steps:

[0024] (1) Screening of broodstock to be induced to spawn: Select healthy female and male fish aged 3 or above, with a male-female ratio of 1:(1.5-2.5), and place them in a broodstock pond for cultivation to obtain broodstock to be induced to spawn;

[0025] (2) Artificial induced spawning: The broodstock to be induced for spawning is injected with the inducing agent using the two - injection method. The first injection is LRH - A3, and the second injection is LRH - A3+HCG. The injection dose for male fish is half of that for female fish, and the injection interval is 11h - 13h;

[0026] (3) Artificial insemination: 24 hours after the injection of the inducing agent, female fish with mature ovarian development are selected and the abdomen is pressed to manually squeeze out eggs, and dry insemination is used to obtain fertilized eggs;

[0027] (4) Screening and hatching of fertilized eggs: The fertilized eggs are transferred to a conical hatching barrel, and high - quality fertilized eggs floating on the water surface are collected with a 60 - mesh silk screen and placed in the hatching pond for hatching.

[0028] In some preferred embodiments, the broodstock pond in step (1) is a circular cement pond with a water depth of 50 - 80 cm and an area of 15 - 20 m 2 , equipped with a temperature control, lighting, oxygenation and inlet - outlet water facility system; the water inlet forms a 45° angle with the pool wall along the same direction, and the drain outlet is located in the center of the pool bottom; the dissolved oxygen in the water body is 5 - 8 mg / L, the salinity is 24 - 28, the pH is 7.5 - 8.2, the water is changed twice a day, the water change volume is 200%, and the sewage is sucked once in the morning and once in the evening. Its technical effect is that by simulating the natural living environment of Hapalogenys mucronatus in the broodstock pond, the vitality of the broodstock is improved, and a water flow is formed by the water inlet with a 45° angle to the pool wall. The water flow stimulates the lateral line organs of the broodstock to synchronize the gonadal development of male and female broodstock and induce the female fish to lay eggs.

[0029] In some preferred embodiments, the injection dose of LRH - A3 for the first injection of female fish in step (2) is 0.5 - 1.5 μg / kg; the injection dose of LRH - A3 for the second injection of female fish is 1 - 3 μg / kg, and the injection dose of HCG is 50 - 150 IU / kg. Its technical effect is that the two - injection hormone injection method is used for artificial induced spawning. The first injection of low - dose LRH - A3 simulates the reproductive signal of Hapalogenys mucronatus in the natural environment, inducing the pituitary gland cells of the broodstock to produce endogenous gonadotropin; the additional LRH - A3+HCG in the second injection can continuously stimulate the pituitary gland cells, and the exogenous HCG directly acts on the gonads, inducing the maturation of female fish oocytes and stimulating ovulation; because the gonads of male fish have a high response degree to sex hormones, half of the sex hormone dose of female fish is used for male fish to avoid premature maturation of the testis leading to premature senescence of sperm; compared with the prior art, this technical scheme significantly reduces the use dose of LRH - A3 and HCG, reduces the cost of artificial induced spawning, and effectively slows down the impact of high - dose sex hormones on the normal physiological functions of the gonadal tissues of the broodstock while ensuring the efficiency of induced spawning.

[0030] In some preferred embodiments, when injecting oxytocin in step (2), the head of the broodstock is wrapped with a wet towel soaked in seawater, and thoracic injection is performed. The injection position is the base of the pectoral fin of the broodstock. The technical effect is as follows: Wrapping the broodstock with a wet towel simulates the water pressure and blocks the light, inhibits the struggle of the broodstock, maintains the moist state of the gills of the broodstock, reduces the difficulty of the injection operation, and reduces the damage to the broodstock caused by being separated from the water environment.

[0031] In some preferred embodiments, the water temperature of the broodstock pond is 23°C after injecting the first dose of oxytocin in step (2); the water temperature of the broodstock pond is adjusted to 25°C after injecting the second dose; the breeding workshop is kept quiet, and feeding is stopped simultaneously. The technical effect is as follows: Promote the oxytocin effect through water temperature regulation. After injecting the first dose, simulate the starting temperature of the natural reproduction of female Hapalogenys mucronatus in the broodstock pond environment, and enhance the effect of LRH-A3 in promoting the pituitary gland to synthesize endogenous gonadotropin in the broodstock; after injecting the second dose, raise the temperature to the optimal temperature for ovulation of female Hapalogenys mucronatus, enhance the metabolic intensity of the female fish, and enhance the synergistic effect of HCG and endogenous gonadotropin, prompting the germinal vesicle of the oocytes of the female fish to rupture and transform into mature eggs; the simultaneous implementation of the silent management in the workshop effectively inhibits the secretion of stress cortisol in the broodstock, reduces the stress response, ensures the normal conduction of the sex hormone signaling pathway, and combines the feeding cessation strategy to avoid premature spawning of female fish due to feeding activities, thereby increasing the yield of mature eggs of female fish in artificial insemination operations.

[0032] In some preferred embodiments, the criteria for screening female fish with mature ovarian development in (3) are that when touching the abdomen of the female fish by hand, it feels soft; the eggs are felt to be separated and flowing in the ovary; the genital pore protrudes after gently pressing the abdomen of the female fish.

[0033] In some more preferred embodiments, the operation cycle for screening female fish with mature ovarian development in (3) is once every 30 - 60 minutes. The technical effect is as follows: Through high-frequency screening of female fish, the mature state of the eggs of female fish is monitored in real time, and eggs in the best fertilization window period are obtained through artificial egg extrusion, which can ensure the potential of embryonic development and improve the fertilization rate; timely screen out female fish to be spawned, reduce the inhibition of pheromone release by dominant female fish on the spawning of other female fish, and accelerate the egg maturation progress of the remaining female fish; timely discharge the eggs in the ovary of female fish to avoid the production of overripe eggs, thereby improving the quality of fertilized eggs in a single batch.

[0034] In some preferred embodiments, the dry fertilization operation method in (3) is to dry the abdomen of the female fish with a clean towel, gently squeeze the abdomen by hand, squeeze the mature eggs into a washbasin that is clean, dry and avoids strong light, squeeze the semen onto the fish eggs, gently stir evenly with a feather for 30 s, add sand-filtered seawater, continue to stir evenly for 30 s, and then slowly add sand-filtered seawater and stir evenly for 1 min to make the eggs and sperm fully combine. Let it stand for 4 - 5 min, then filter out the excess semen with a 60-mesh silk screen, and wash the eggs with seawater 2 - 3 times. The technical effect is that: since the lifespan of the sperm of Hapalogenys mucronatus is only about 7 minutes after being activated by seawater, in this technical solution, the eggs and sperm are added to a dry washbasin successively and then sand-filtered seawater is added to prevent the sperm from being prematurely activated by seawater and affecting the sperm activity; the gentle stirring with a feather promotes the uniform contact of sperm and eggs and avoids mechanical damage to the egg membrane structure; adding sand-filtered seawater in stages simulates the osmotic pressure change of natural fertilization and promotes the combination of eggs and sperm; the precise 4 - 5-minute standing period ensures that the fertilization pores of the eggs are completely closed, and the combination of 60-mesh silk screen filtration and seawater egg washing effectively removes the excess sperm and eliminates the toxic effect of sperm metabolites on the embryo.

[0035] In some preferred embodiments, in the dry fertilization operation in (3), the volume ratio of eggs, semen and sand-filtered seawater is 1000:(1 - 2):2500; the semen is collected from 2 - 3 male fish. The technical effect is that: through the volume ratio, it is ensured that the number of sperm that can be paired with a unit of eggs is within the most suitable range, which can not only meet the competition and screening requirements of eggs for sperm but also avoid the cumulative toxicity of reactive oxygen species caused by excessive sperm metabolism; using the mixed semen of 2 - 3 male fish can reduce the change in fertilization rate caused by the difference in sperm activity between male individuals, improve the operation stability of this technical solution, and adapt to artificial large-scale farming.

[0036] In some preferred embodiments, the time for collecting high-quality fertilized eggs in step (4) is 5 - 7 hours after the fertilized eggs are put into the hatching barrel. After the high-quality fertilized eggs are placed in the hatching pond for 28 - 32 hours, newly hatched larvae are obtained. The high-quality fertilized eggs at the low blastula stage float on the water surface, while the unfertilized eggs and the eggs with embryonic development stopped will sink to the bottom of the barrel or suspend in the water body, and the high-quality fertilized eggs floating on the water surface are separated with a 60-mesh silk screen fishing net. The technical effect is that: the fertilized eggs developed for 6 h are at the low blastula stage, and they float up due to the expansion of the perivitelline space and the accumulation of lipid metabolites, while the dormant eggs sink due to the sedimentation of yolk substances. Combined with the physical separation of a 60-mesh silk screen, more than 98% of abnormal eggs can be removed, and the bacteria attached to the egg membrane surface can be synchronously removed, improving the hatching rate of fertilized eggs. Specific embodiments

[0038] Embodiment

[0039] (1) Screening of broodstock to be induced to spawn:

[0040] Select 10 three-year-old female fish and 20 three-year-old male fish with normal body color, good health and no deformities, and place them in a circular broodstock pond with a water depth of 50 cm and an area of 20 m 2 , equipped with a temperature control, lighting, oxygenation and water inlet and outlet facility system for cultivation; the water inlet of the broodstock pond forms a 45° angle with the pool wall along the same direction, and the drain outlet is located in the center of the pool bottom; the dissolved oxygen in the water body is 8 mg / L, the salinity is 28, and the pH is 7.5. Change the water twice a day, with a water change volume of 200%, and suck the sewage once in the morning and once in the evening.

[0041] (2) Artificial induction of spawning:

[0042] When the abdomen of the female fish bulges, start the work of inducing spawning. When injecting the inducing hormone, wrap the head of the broodstock with a wet towel soaked in seawater, and use thoracic injection. Insert the needle when the syringe forms a 45° angle with the base of the pectoral fin of the broodstock, and the injection depth is 6 - 8 mm; for the first injection of the female fish, inject 1 μg / kg of LRH-A3, and for the second injection of the female fish, inject 2 μg / kg of LRH-A3 + 100 IU / kg of HCG. The injection amount for the male fish is half of that of the female fish, and the injection interval is 12 h. After the first injection of the inducing hormone, the water temperature in the broodstock pond is 23°C; after the second injection, the water temperature in the broodstock pond is adjusted to 25°C; keep the breeding workshop quiet and stop feeding the bait at the same time.

[0043] (3) Artificial insemination:

[0044] After 24 h of inducing spawning, screen the female fish with mature ovarian development for artificial abdominal compression to squeeze out eggs every 40 min. The screening criteria for female fish with mature ovarian development are that when stroking the abdomen of the female fish by hand, it feels soft, and at the same time, the eggs can be felt separating and flowing in the ovary, and the genital pore of the female fish can protrude when gently pressing the abdomen. After screening, dry the abdomen of the female fish with a clean towel, gently squeeze the abdomen by hand, squeeze the mature eggs into a washbasin that is clean, dry and avoids strong light irradiation, and then squeeze the semen onto the fish eggs. To improve the fertilization rate, squeeze the semen of 2 male fish into each batch. For every about 1000 mL of eggs as a batch, the amount of semen added is 2 mL. Stir gently with a feather evenly for 30 s, add 500 mL of sand-filtered seawater, continue to stir evenly for 30 s, and then slowly add 2000 mL of sand-filtered seawater and stir evenly for 1 min to make the eggs and sperm fully combine. Let it stand for 5 min, and then filter out the excess semen with a 60-mesh silk screen and wash the eggs with seawater 3 times.

[0045] (4) Screening and hatching of fertilized eggs:

[0046] Transfer the obtained fertilized eggs into a conical hatching bucket. Place an aeration stone at the bottom of the bucket to keep the fertilized eggs tumbling up and down continuously, avoiding the accumulation of fertilized eggs at the bottom of the pond. Control the hatching water temperature at 23°C, salinity at 25, pH at 7.5, and dissolved oxygen at 8 mg / L. After the fertilized eggs are hatched in the conical bucket for 6 hours and reach the low blastula stage, stop aerating the aeration stone. After standing for 10 minutes, the high-quality fertilized eggs will float on the water surface, while the unfertilized eggs and the eggs with arrested embryonic development will sink to the bottom of the bucket or suspend in the water body. Gently separate the high-quality fertilized eggs floating on the water surface with a 60-mesh silk screen fishing net; transfer the separated high-quality fertilized eggs into a hatching pond with a length of 8 m, a width of 4 m, and a water depth of 1 m. Distribute 20 aeration stones evenly at the bottom of the pond and aerate slightly. The egg density in the water body is 5000 eggs / m3, the hatching water temperature is 23°C, the salinity is 24, and the pH is 7.5. After another 30 hours, newly hatched larvae can be obtained. Continuously culture the obtained newly hatched larvae to obtain the Figure 1 juvenile Hapalogenys mucronatus as shown

[0047] Comparative Example 1

[0048] (1) Selection of broodstock:

[0049] Select 150 three-year-old broodstock with a body weight of over 200 g and put them into a circular broodstock pond with a water depth of 50 cm and an area of 20 m 2 equipped with a temperature control, lighting, oxygenation, and water inlet and outlet facility system for cultivation. Among them, there are 100 female fish and 50 male fish.

[0050] (2) Natural fertilization:

[0051] Through environmental regulation measures, promote the natural development of the gonads of Hapalogenys mucronatus to sexual maturity. Wait until the male and female fish show chasing behavior and let them mate naturally to produce fertilized eggs.

[0052] (3) Collection and hatching of fertilized eggs:

[0053] Collect the fertilized eggs into the egg box by the overflow method every evening; the next day, collect the fertilized eggs floating on the water surface with a silk screen net. Add the fertilized eggs in the egg box and the silk screen net into a plastic bucket filled with seawater, aerate slightly, collect the normally developing fertilized eggs floating on the surface with a silk screen net, and discard the bad eggs at the bottom layer. Distribute the fertilized eggs evenly in the circular hatching bucket. The hatching water temperature in the circular hatching bucket is 21°C, the salinity is 25, the pH value is 7.5, and aerate slightly. One day later, the fertilized eggs will hatch successively.

[0054] Comparative Example 2

[0055] (1) Selection of broodstock:

[0056] Select 10 three-year-old female fish with normal body color, good health, and no deformities and 20 three-year-old male fish, and put them into a circular broodstock pond with a water depth of 50 cm and an area of 20 m 2 equipped with a temperature control, lighting, oxygenation, and water inlet and outlet facility system for cultivation.

[0057] (2) Artificial induced spawning:

[0058] When injecting the oxytocin, wrap the head of the broodstock with a wet towel soaked in seawater, and take intrathoracic injection. When the syringe forms a 45° angle with the base of the pectoral fin of the broodstock, insert the needle, and the injection depth is 6 - 8 mm; the female fish is injected with 0.5 μg / kg of LRH-A2 for induced spawning, and the injection amount of the male fish is half of that of the female fish. Keep the breeding workshop quiet and stop feeding the bait at the same time.

[0059] (3) Natural fertilization:

[0060] Combined with environmental regulation measures, promote the natural development of the gonads of Hapalogenys mucronatus to sexual maturity. When chasing behavior appears between male and female fish, let them mate naturally to produce fertilized eggs.

[0061] (4) Collection and hatching of fertilized eggs:

[0062] Collect the fertilized eggs into the egg box by the overflow method every evening; the next day, collect the fertilized eggs floating on the water surface with a sieve net. Add the fertilized eggs in the egg box and the sieve net into a plastic bucket filled with seawater, slightly aerate, collect the normally developing fertilized eggs floating on the surface with a sieve net, and discard the bad eggs at the bottom layer; evenly distribute the normal fertilized eggs into a round hatching bucket. The hatching water temperature in the round hatching bucket is 21 - 25 °C, the salinity is 25 - 30‰, the pH value is 7.5 - 8.0, slightly aerate, and the fertilized eggs will hatch one after another after one day.

[0063] Comparative Example 3

[0064] The difference from Comparative Example 2 is that in step (2), the dose of LRH-A2 used for artificial induced spawning of female fish is 1 μg / kg.

[0065] Comparative Example 4

[0066] The difference from Comparative Example 2 is that in step (2), the oxytocin used for artificial induced spawning of female fish is HCG 50 IU / kg + LRH-A2 0.5 μg / kg.

[0067] Comparative Example 5

[0068] The difference from Comparative Example 2 is that in step (2), the oxytocin used for artificial induced spawning of female fish is HCG 100 IU / kg + LRH-A2 1 μg / kg.

[0069] Comparative Example 6

[0070] The difference from Comparative Example 2 is that in step (2), the oxytocin used for artificial induced spawning of female fish is LRH-A3 0.5 μg / kg.

[0071] Comparative Example 7

[0072] The difference from Comparative Example 2 is that the oxytocin used for artificial induction of female fish in step (2) is 1 μg / kg of LRH-A3.

[0073] Comparative Example 8

[0074] The difference from Comparative Example 2 is that the oxytocin used for artificial induction of female fish in step (2) is 50 IU / kg of HCG + 0.5 μg / kg of LRH-A3.

[0075] Comparative Example 9

[0076] The difference from Comparative Example 2 is that the oxytocin used for artificial induction of female fish in step (2) is 100 IU / kg of HCG + 1.0 μg / kg of LRH-A3.

[0077] Comparative Example 10

[0078] (1) Screening of broodstock to be induced:

[0079] Select 10 three-year-old female fish and 20 three-year-old male fish with normal body color, good health, and no deformities, and put them into a circular broodstock pond with a water depth of 50 cm and an area of 20 m 2 , equipped with a temperature control, lighting, oxygenation, and water inlet and outlet facility system for cultivation.

[0080] (2) Artificial induction:

[0081] When the abdomen of the female fish bulges, perform the induction work. When injecting oxytocin, wrap the head of the broodstock with a wet towel soaked in seawater, and perform thoracic injection. Insert the needle at a 45° angle to the base of the pectoral fin of the broodstock, and the injection depth is 6 - 8 mm; inject 1 μg / kg of LRH-A2 into the female fish, and the injection amount for the male fish is half of that of the female fish. Keep the breeding workshop quiet and stop feeding the bait at the same time.

[0082] (3) Artificial insemination:

[0083] After 24 hours of induction, screen the female fish with mature ovarian development every 40 minutes for artificial abdominal compression to squeeze out eggs. The screening criteria for female fish with mature ovarian development are that when touching the abdomen of the female fish by hand, it feels soft, and at the same time, the eggs can be felt separating and flowing in the ovary, and the genital pore of the female fish can protrude when gently pressing the abdomen. After screening, dry the abdomen of the female fish with a clean towel, gently squeeze the abdomen by hand, squeeze the mature eggs into a washbasin that is clean, dry, and avoids strong light irradiation, then squeeze the semen onto the fish eggs. To improve the fertilization rate, squeeze the semen of 2 male fish into each batch, about 1000 mL of eggs is a batch, and the amount of semen added is 2 mL. Gently stir evenly with a feather for 30 s, add 500 mL of sand-filtered seawater, continue to stir evenly for 30 s, then slowly add 2000 mL of sand-filtered seawater, stir evenly for 1 min to make the eggs and sperm fully combine, let it stand for 5 min, then filter off the excess semen with a 60-mesh silk screen, and wash the eggs 3 times with seawater.

[0084] (4) Screening and hatching fertilized eggs:

[0085] Transfer the obtained fertilized eggs into a conical hatching bucket, place an aeration stone at the bottom of the bucket to make the fertilized eggs roll up and down continuously, avoid the accumulation of fertilized eggs at the bottom of the pond, control the hatching water temperature at 23°C, salinity at 25, pH at 7.5, and dissolved oxygen at 8 mg / L. After 6 hours of hatching in the conical bucket, the fertilized eggs reach the low blastula stage, stop aerating the aeration stone, and after standing for 10 minutes, the high-quality fertilized eggs float on the water surface, while the unfertilized eggs and the eggs with embryonic development stopped will sink to the bottom of the bucket or suspend in the water body. Gently separate the high-quality fertilized eggs floating on the water surface with a 60-mesh silk screen fishing net; transfer the separated high-quality fertilized eggs into a hatching pond with a length of 8 m, a width of 4 m, and a water depth of 1 m. There are 20 aeration stones evenly distributed at the bottom of the pond, with slightly aerated. The egg density in the water body is 5000 - 7000 eggs / m³, the hatching water temperature is 23°C, salinity is 26, pH is 7.5, and dissolved oxygen is 6 mg / L. After another 30 hours, newly hatched larvae can be obtained.

[0086] Comparative Example 11

[0087] The difference from Comparative Example 10 is that in step (2), the artificial induction of the female fish uses the two - injection method to inject LRH - A2. The first injection is LRH - A2 at 1.0 μg / kg, and the second injection is LRH - A2 at 2.0 μg / kg, and the time interval between the two injections is 12 hours.

[0088] Comparative Example 12

[0089] The difference from Comparative Example 10 is that in step (2), the oxytocin used for the artificial induction of the female fish is HCG 100 IU / kg + LRH - A2 1 μg / kg.

[0090] Comparative Example 13

[0091] The difference from Comparative Example 12 is that in step (2), the artificial induction of the female fish uses the two - injection method to inject HCG + LRH - A2. The first injection is LRH - A2 at 1.0 μg / kg, and the second injection is HCG 100 IU / kg + LRH - A2 1 μg / kg, and the time interval between the two injections is 12 hours.

[0092] Comparative Example 14

[0093] The difference from Comparative Example 10 is that in step (2), the oxytocin used for the artificial induction of the female fish is LRH - A3 1 μg / kg.

[0094] Comparative Example 15

[0095] The difference from Comparative Example 14 is that in step (2), the artificial induction of labor of female fish is performed by injecting LRH-A3 with two injections, the first injection is LRH-A3 1.0 μg / kg and the second injection is LRH-A3 2.0 μg / kg, and the time interval between the two injections is 12 hours.

[0096] Comparative Example 16

[0097] The difference from Comparative Example 10 is that the oxytocin used for artificial induction of labor in female fish in step (2) is HCG 100 IU / kg + LRH-A3 1.0 μg / kg.

[0098] Table 1 Operation variable comparison table

[0099] Test example

[0100] The fertilized eggs of the horizontal banded bream were obtained by using the methods of the embodiment and the comparative examples, and the egg laying amount, fertilization rate and hatching rate of the horizontal banded bream were statistically analyzed. The statistical results are shown in Table 2.

[0101] Table 2

[0102] The experimental results show that the present invention significantly improves the artificial breeding efficiency of the horizontal banded bream by systematically integrating reproductive regulation strategies with standardized artificial insemination processes. In the embodiment using the technical solution of the present invention, the single egg-laying capacity of female fish reaches 6.9×105 eggs / tail, the fertilization rate reaches 63-72%, and the hatching rate reaches 95%; it can be seen from the embodiments and comparative examples 1-9 that the use of oxytocin can increase the egg-laying capacity of the horizontal banded bream, however, compared with the preferred embodiment of the present invention, its fertilization rate and hatching rate still have a significant gap; comparative examples 10-16 use artificial induction combined with artificial insemination technology, and its fertilization rate and hatching rate are significantly improved compared with the natural fertilization group (comparative examples 1-9), but are still significantly lower than the preferred embodiment of the present invention. In summary, the present invention uses precise control technology of the spawning cycle to synchronize the hatching time of fertilized eggs in the same batch, laying the foundation for large-scale breeding of fry; and optimizes the timing of hormone injection and the mixing ratio of sperm and eggs, while reducing stress damage to parent fish, increasing the output of fertilized eggs per parent fish, successfully overcoming technical problems such as insufficient male semen and asynchronous development of males and females, and providing key technical support for establishing an artificial breeding system for cross-banded sea bream that is independent of wild resources.

[0103] The raw materials and equipment used in the present invention are all common raw materials and equipment in the art unless otherwise specified; the methods used in the present invention are all conventional methods in the art unless otherwise specified. The above description is only a preferred embodiment of the present invention and does not impose any limitation on the present invention. Any simple modification, change, and equivalent transformation made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus, characterized in that, It includes the following steps: (1) Select healthy female and male fish aged 3 or above, with a female-male ratio of 1:1.5 - 2.5, and put them into the broodstock pond for cultivation to obtain broodstock to be induced to spawn. (2) The broodstock to be induced to spawn is injected with an ovulation-inducing agent using the two-shot method. For the first shot, LRH-A3 is injected, and for the second shot, LRH-A3 + HCG is injected. The injection dose for male fish is half of that for female fish, and the injection interval is 11h - 13h. (3) After 24h of injecting the ovulation-inducing agent, select female fish with mature ovarian development, press their abdomens to perform artificial egg extrusion, and obtain fertilized eggs by dry fertilization. (4) Transfer the fertilized eggs into a conical hatching barrel, and use a 60-mesh silk screen to collect high-quality fertilized eggs floating on the water surface and place them in the hatching pond for hatching.

2. An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus according to claim 1, characterized in that, In step (1), the broodstock pond is a circular cement pond with a water depth of 50-80 cm and an area of 15-20 m 2 , equipped with a temperature control, lighting, oxygenation and inlet and outlet water facilities system; the water inlet forms a 45° angle with the pool wall along the same direction, and the drain outlet is located at the center of the pool bottom; the dissolved oxygen in the water body is 5-8 mg / L, the salinity is 24-28, the pH is 7.5-8.2, the water is changed twice a day, the water change volume is 200%, and the sewage is sucked once in the morning and once in the evening.

3. An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus according to claim 1, characterized in that, In step (2), the injection dose of LRH-A3 for the first shot of female fish is 0.5 - 1.5 μg / kg; the injection dose of LRH-A3 for the second shot of female fish is 1 - 3 μg / kg, and the injection dose of HCG is 50 - 150 IU / kg.

4. An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus according to claim 1 or 3, characterized in that, In step (2), when injecting the ovulation-inducing agent, wrap the head of the broodstock with a wet towel soaked in seawater, and perform thoracic injection. The injection position is the base of the pectoral fin of the broodstock.

5. An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus according to claim 1 or 3, characterized in that, In step (2), after injecting the first shot of the ovulation-inducing agent, the water temperature in the broodstock pond is 23°C, and a sunshade net is covered on the broodstock pond; after injecting the second shot, the water temperature in the broodstock pond is adjusted to 25°C, and the sunshade net is still covered; keep the breeding workshop quiet, and at the same time stop feeding the bait.

6. The artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus according to claim 1, wherein In step (3), the criteria for selecting female fish with mature ovarian development are that when touching the abdomen of the female fish by hand, it feels soft; the eggs are felt to be separated and flowing in the ovary; when gently pressing the abdomen of the female fish, the genital pore protrudes.

7. An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus according to claim 1 or 6, characterized in that In step (3), the operation cycle for screening ovulating female fish is once every 30 - 60 minutes.

8. An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus according to claim 1, characterized in that, In step (3), the dry fertilization operation is to dry the abdomen of the female fish with a clean towel, gently squeeze the abdomen of the female fish, squeeze the mature eggs into a washbasin that is clean, dry and avoids strong light, squeeze the semen onto the fish eggs, gently stir evenly with a feather for 30s, add sand-filtered seawater, continue to stir evenly for 30s, then slowly add sand-filtered seawater and stir evenly for 1min to make the eggs and sperm fully combine, let it stand for 4 - 5min, then filter off the excess semen with a 60-mesh silk screen, and wash the eggs with seawater 2 - 3 times.

9. An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus according to claim 1 or 8, characterized in that, In the dry fertilization operation in step (3), the volume ratio of eggs, semen and sand-filtered seawater is 1000:1 - 2:2500; the semen is collected from 2 - 3 male fish.

10. An artificial insemination method for improving the fertilization rate of Hapalogenys mucronatus according to claim 1, characterized in that, In step (4), the time for collecting high-quality fertilized eggs is 5 - 7 hours after the fertilized eggs are put into the hatching barrel; after the high-quality fertilized eggs are placed in the hatching pond for 28 - 32 hours, newly hatched larvae are obtained.

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