Method for regulating out-of-season breeding of large yellow croakers bred in autumn

Abstract

The invention discloses a method for regulating out-of-season breeding of large yellow croakers bred in autumn. The method is characterized in that spawning in spring and summer is achieved through a three-stage technical system of biological safety prevention and control, precise environment regulation and control and reproductive physiological intervention. The method specifically comprises the following steps: 1) detecting trypanosomes; 2) parent fish screening; (3) parent fish rearing; 4) artificial propagation; 5) artificial incubation; and 6) seedling cultivation. According to the method, the out-of-season spring and summer breeding technology breakthrough of breeding the large yellow croaker in autumn is successfully achieved through nutrient enrichment, accurate temperature parameter regulation and control, gradual illumination regulation and reasonable hormone treatment. According to the technology, an adjustable reproduction synchronization scheme is provided for cross breeding between a spring reproduction group and an autumn reproduction group, and the technology has important application value for germplasm innovation of the large yellow croaker.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a method for regulating the off-season reproduction of large yellow croaker that reproduces in autumn. Background Technology

[0002] Many fish exhibit typical seasonal patterns in their reproductive activities. Under natural conditions, gonadal development, gamete proliferation and maturation, spawning, and embryonic development occur only during specific breeding seasons. Large yellow croaker (… Larimichthys crocea Large yellow croaker is one of the main fish species in my country's marine aquaculture, occupying an important position in the fishery economy of major producing areas such as Fujian and Zhejiang. Its annual aquaculture output has long ranked first among marine aquaculture fish, making its economic value extremely prominent. The breeding season of large yellow croaker is mainly concentrated in spring, summer, and autumn. Among them, the East China Sea large yellow croaker mainly breeds in spring and summer, while the South China Sea large yellow croaker mostly breeds in autumn. The peak breeding season in spring and summer usually occurs from April to June, while the peak breeding season in autumn is concentrated from October to November.

[0003] A search revealed no publicly available reports of artificially controlling the breeding of large yellow croaker in autumn to achieve off-season spring breeding. Traditionally, autumn-bred large yellow croaker fry must endure the low temperatures of winter, leading to a series of technical challenges. When water temperatures drop below 14℃, the fry's feeding decreases significantly; as the temperature falls further, they cease feeding entirely. During severe weather events such as cold waves, the mortality rate of small fry in outdoor environments like net cages rises sharply. These factors severely restrict the expansion and economic benefits of autumn-bred large yellow croaker farming. Overcoming the technical limitations of off-season breeding in autumn and achieving spring / summer breeding, synchronizing the outdoor rearing time of fry with the optimal water temperature (18-28℃), can significantly improve fry survival rates, effectively reduce farming costs, and substantially enhance farming efficiency and economic benefits. Furthermore, the successful application of this technology can facilitate crossbreeding between spring-bred and autumn-bred large yellow croaker populations, creating new varieties, which is of great significance for the sustainable development of the large yellow croaker breeding industry and the entire aquaculture sector.

[0004] Trypanosomiasis is one of the most destructive parasitic diseases in the large yellow croaker aquaculture industry. Since its initial outbreak in Fujian in September 2023, the disease has rapidly spread to several major aquaculture areas in Fujian, and in 2024, it affected major aquaculture areas in Zhejiang, including Taizhou, Wenzhou, Ningbo, and Zhoushan. During the peak disease season from June to September, the mortality rate of infected fry can exceed 60%. Trypanosomiasis can infect large yellow croaker at different growth stages, including juveniles, medium-sized fish, and adults. Vertical transmission due to infection of broodstock is a weak link in the prevention and control system. Summary of the Invention

[0005] In view of the problems in the prior art, the present application is designed to provide a method for regulating off-season reproduction of autumn-reproducing Pseudosciaena crocea, which is specifically implemented by the following technical solutions: Step one: Trypanosoma detection Before selecting the parent fish, Trypanosoma detection should be performed to ensure that the parent fish is not infected with Trypanosoma. The specific operation is as follows: Blood smear preparation: After the Pseudosciaena crocea is anesthetized with eugenol, blood is taken from the tail vein with a sterile syringe containing 3.0% trisodium citrate anticoagulant at a ratio of 1:4 of anticoagulant to blood volume. After blood collection, smear preparation is immediately performed. Take a clean cover glass as a push piece, and drop 1-2 drops of blood on one end of the glass slide. Gently contact the push piece with the glass slide at an angle of 30-45°, and then push the push piece forward at a steady and uniform speed to form a uniform and thin blood film on the glass slide.

[0006] Staining and microscopy: After the smear is completed, the glass slide is placed in a well-ventilated place and naturally dried in the air. The dried blood smear can be stained with Giemsa staining or Wright staining or Wright-Giemsa rapid staining solution. When staining, the staining solution should evenly cover the blood film. After staining, rinse the glass slide from one end with slow running water for 30 seconds. The water flow should not be too fast to prevent the blood film from being washed away. After rinsing, the glass slide is dried vertically. The dried blood smear can be observed under a microscope.

[0007] Trypanosoma infection determination standard: In the blood smear of healthy Pseudosciaena crocea, only red blood cells and white blood cells can be observed under a microscope oil lens. If typical willow or wave-shaped trypanosomes, long flagella and elliptical kinetoplasts and other characteristic structures are observed, it is determined that the fish is infected with Trypanosoma.

[0008] Step two: Parent fish screening Every year from December to early January, select Pseudosciaena crocea that are 2 winters old, have a robust body, no damage to the body surface, and good development as parent fish. Preferably, select female fish with a body weight of more than 550g and male fish with a body weight of more than 450g. The gonads are required to be in phase II, and 3-5 fish can be dissected to observe the gonadal development.

[0009] The parent fish without Trypanosoma infection is transferred to the cultivation pool. The cultivation pool can be selected from indoor cement pool, circular glass steel water tank or PP circular breeding barrel as the parent fish cultivation pool, and the cultivation pool has a diameter of more than 3m and a water depth of 1.5-2m. The cultivation pool is equipped with a temperature control system, a water inlet and outlet system, an oxygenation device and a light adjustment device.

[0010] After the parent fish is temporarily raised for one week, the second Trypanosoma detection is performed, 15 parent fish are randomly selected from the temporarily raised parent fish, and the second Trypanosoma detection (blood smear preparation, staining and microscopy) is performed according to the method described in step one to ensure that there is no Trypanosoma infection.

[0011] Step three: broodstock cultivation Temperature stimulation of gonadal development: After the end of the temporary cultivation, gradually increase the water temperature by 1°C every two days to stimulate the initiation of gonadal development in autumn-spawning P. olivaceus. When the temperature reaches 23-25°C, stop increasing the temperature and maintain a stable water temperature.

[0012] Light regulation: From the middle to late January, simulate the natural light and temperature cycle in spring and summer by gradually extending the light time using artificial light sources. Increase the light time by 1 hour per day until the total light time reaches 16-20 hours per day. LED lights can be installed above the cultivation pond. The light intensity should be controlled at 450-600 lx.

[0013] Nutritional enhancement: Feed high-quality P. olivaceus special compound feed with a protein content of ≥45% and a fat content of 8-12%. Add 5%-8% of Chlorella powder to the compound feed to provide unsaturated fatty acids. Feed 2-3 times a day, and adjust the feeding amount based on the fish weight and feeding conditions, generally 1-3% of the body weight. In addition, regularly supplement vitamins and minerals by feeding compound feed with added vitamins and minerals every 10 days.

[0014] Water quality conditions during broodstock cultivation: Dissolved oxygen content ≥6 mg / L, salinity 21-30‰, pH 7.85-8.35. Check the water quality once a week.

[0015] Step four: artificial reproduction Broodstock examination: In the later stage of broodstock cultivation, closely monitor gonadal development. When the abdomen of female fish is significantly swollen, it indicates that the eggs have developed to stage IV, and regular examination of gonadal development is required every 7 days. When the abdomen of female fish is very full and the cloaca is obviously open, use a pipette to extract the eggs for examination. If the eggs are uniform in size and easy to separate, it indicates that the eggs have developed to the end of stage IV. Lightly pressing the abdomen of male fish causes sperm to flow out, and the sperm quickly disperses in the water, indicating that the sperm has matured. Artificial induction of spawning can be performed in a timely manner.

[0016] Artificial induction of spawning: Use exogenous hormone injection to induce spawning. The exogenous hormone injection dose is luteinizing hormone-releasing hormone analogue (LRH-A3), and the injection site is the base of the pectoral fin. The dose for female fish is 2-5 μg / kg of body weight, and the dose for male fish is half of that. Single or double injections can be performed based on the degree of gonadal maturation. For double injections, the first injection is 1 / 2 of the total dose, and the second injection is given 24-48 hours later. Place the P. olivaceus injected with the induction agent in the spawning tank at a female to male ratio of 1-2:1, and allow them to complete spawning and sperm release. The spawning tank should be kept in a weak light or dark state to avoid direct sunlight, and kept quiet. At the same time, increase the flow rate of the circulating water to promote natural spawning.

[0017] Step five: artificial incubation Within 3 hours after spawning, the fertilized eggs floating on the surface or upper layer of the water body are collected by using a 80-mesh net and are put into a hatching tank for hatching. The air inflation method or the flowing water method can be used for hatching. The oxygenation or the flowing water speed of the hatching tank is adjusted so that the fertilized eggs can be suspended and slowly stirred in the water body of the hatching tank. The development of the fish eggs is checked in time during the hatching process, the embryo development process is observed, and the dead eggs and unfertilized eggs are removed in time until the membrane is out. The water for hatching must be seawater which is treated by sedimentation, filtration and disinfection, and the water quality is required to be clear and free of heavy metal pollution, and the water temperature is required to be between 23-25 DEG C.

[0018] Step six: fry cultivation After 30-35 hours, the fry hatches out of the membrane, usually on the 3rd-4th day, the yolk sac is completely absorbed, and the larvae start to feed. After the larvae start to feed, the larvae are fed with the opening food Brachionus plicatilis, and the feeding density is 10-15 pieces / mL; the rotifer is nutritionally enhanced by Chlorella, and the Chlorella enhancement concentration is generally 18-20 million pieces / mL; before the second feeding each day, the remaining rotifers in the cultivation tank are counted and the rotifers are supplemented. After 8 days, Artemia nauplii (brine shrimp) are fed, and the feeding amount is about 100-200 pieces of Artemia nauplii per fry per day, and the feeding is performed twice a day, and the specific feeding amount is adjusted according to the feeding condition, and it is appropriate that the larvae basically finish feeding within 1.5-2 hours after feeding. After 11 days, the larvae are fed with Artemia nauplii and micro-particle compound feed, and the feeding is performed twice a day; after 15 days, the feeding amount of the Artemia nauplii is reduced; after 20 days, the larvae are fed with the micro-particle compound feed alone, and the feeding is performed 3-4 times a day; and the feeding amount is appropriate when the larvae feed actively and disperse naturally after being full.

[0019] Before the parent fish cultivation tank, the hatching tank and the fry cultivation tank are used, the parent fish cultivation tank, the hatching tank and the fry cultivation tank need to be strictly disinfected with 15-20 ppm potassium permanganate solution, and then washed with clean seawater to remove the residual disinfectant, so as to avoid the harm to the parent fish, the fish eggs and the fry.

[0020] The large yellow croaker is sensitive to sound, especially the sound of 100-600 Hz, and during the cultivation of the parent fish and the fry, especially during the spawning and the fry cultivation, a quiet environment is created as much as possible to avoid the noise to cause the stress reaction of the fish.

[0021] The present application successfully realizes the anti-season spring and summer breeding technology breakthrough of the large yellow croaker in the autumn breeding through the nutritional enhancement, the accurate temperature parameter control, the gradual light adjustment and the reasonable hormone treatment, and the anti-season breeding plays a very important role in the breeding of the new variety of the large yellow croaker. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a schematic diagram of the multi-factor synergistic regulation of the gonad development in the present application; Figure 2The micrograph (Wright-Giemsa staining) of the blood smear of the healthy large yellow croaker shows that only red blood cells and white blood cells can be seen in the blood smear, the number of red blood cells is large, and the number of white blood cells is small; Figure 3 The micrograph of the micro-morphological characteristics of the trypanosoma (Giemsa staining) is shown in the following figure. Figure 4 The body length growth of the large yellow croaker after being released into a net cage for 2 months is shown in the following figure. DETAILED DESCRIPTION

[0023] The present application will be further described below in combination with the drawings of the specification, so as to better understand the technical solutions.

[0024] The broodstock cultivation pond, the hatching pond and the fry cultivation pond involved in the present application are strictly disinfected with 20 ppm potassium permanganate solution before use, and then clean seawater is injected after being washed clean.

[0025] The schematic diagram of the multi-factor synergistic regulation of the gland development in the present application is shown in the following figure. Figure 1

[0026] Example 1: Blood smear preparation and trypanosoma detection Before the broodstock is transported to the breeding base, 15 large yellow croakers for autumn breeding are randomly selected, anesthetized with eugenol, and 1 ml of blood is extracted from the tail vein using a sterile syringe containing 3.0% (w / v) trisodium citrate anticoagulant at an anticoagulant to blood volume ratio of 1:4. Blood smears are prepared immediately after blood collection. Drop the blood on one end of a clean glass slide, use another cover glass as a pusher, and push it at a 30°-45° angle to the glass slide at a uniform speed to form a uniform thin layer of blood film. The blood smear is placed in a well-ventilated place for natural drying. Dry the blood smear and use Wright-Giemsa rapid staining solution (purchased from Nanjing Jiancheng Biological Engineering Institute) for staining. The specific operation is to add 3-4 drops of staining solution to cover the blood film, stain for 1 minute, then add 6-8 drops of phosphate buffer, and stain for 7 minutes. After staining, rinse the glass slide with slow running water for 30 seconds, and dry it on the rack. Observe under the oil lens of the microscope, and no characteristic structures (lanceolate or wavy body, long flagellum, and oval kinetoplast) of trypanosoma are observed in the blood smears of all the tested fish, indicating no trypanosoma infection.

[0027] Trypanosoma infection determination standard: in the blood smear of a healthy large yellow croaker, only red blood cells and white blood cells can be observed under the oil lens of the microscope (see Figure 2 ). If typical lanceolate or wavy body, long flagellum and oval kinetoplast characteristic structures (see Figure 3 ) are observed, it is determined that the fish is infected with trypanosoma.

[0028] Example 2: Broodstock screening and cultivation ​From early December to January, 120 P. major were selected from a deep-sea net cage culture base. After surface inspection and blood test, 100 P. major with robust constitution, yellow body color, no damage on the body surface, fresh red gills, and no Trypanosoma sp. in the blood were finally selected as broodstock. The weight of female fish was greater than 550 g, and the weight of male fish was greater than 450 g. The broodstock was transported to the breeding base and stocked in two cement broodstock ponds at a stocking density of 2-3 kg / m 3 The broodstock room was equipped with a temperature control system (warm water pool to control water temperature), water intake and discharge system, circulating water filtration system, and oxygenation facilities (high-speed variable frequency air blower to provide air source, nano air disc to diffuse) Broodstock temporary rearing and second Trypanosoma sp. detection: The broodstock was temporarily reared for one week to adapt to the new environment, and the fish were fed with compound feed during this period. After the temporary rearing, 15 broodstock were randomly selected from the temporarily reared broodstock for the second Trypanosoma sp. detection. No characteristic structure of Trypanosoma sp. was observed in the blood smear of the detected fish.

[0029] Temperature stimulation of gonad development: Then the water temperature was gradually increased by 1 degree every 2 days, and the temperature was stopped at 24°C and kept. During the temperature increase, the fish were appropriately fed with compound feed.

[0030] Light regulation: During the later stage of temperature increase, the light time was gradually extended by supplementing light with artificial light source, with an increase of 1 hour per day, and the light intensity was about 550 lx. After the light time reached 18 hours per day, the light time was maintained at 18 hours per day.

[0031] Nutrition enhancement: During the temperature increase, high-quality compound feed was fed, with protein content ≥45%, fat content 10%, and 5% chlorella added. The fish were fed 2-3 times a day, with the feeding amount adjusted according to the fish weight and feeding condition, about 1.5-2% of the body weight. Compound vitamins and compound minerals were added to the feed once every 10 days, and both were commercially available products.

[0032] Example 3: Artificial breeding and hatching Broodstock inspection: The broodstock was inspected for gonad development during the later stage of broodstock cultivation. When the female fish had a significantly swollen abdomen, the cloaca was red and swollen, and the cloaca was significantly open, the eggs were collected for detection. When the egg size was uniform and easy to separate, the male fish was gently pressed on the abdomen, and the sperm flowed out and quickly dispersed in the water. The fish was ready for induced spawning.

[0033] Artificial induced spawning: Induced spawning was performed at the end of March or the beginning of April, and the induced spawning agent LRH-A3 was injected, with a dose of 3 μg / kg for female fish and 1.5 μg / kg for male fish, in two injections, 24 hours apart. After induced spawning, the broodstock was placed in the spawning tank at a ratio of 1.5:1 female to male, and the spawning tank was kept in a state of slight flow. The P. major after induced spawning spawned eggs and sperm.

[0034] Artificial incubation: 2 hours after spawning, stop the flow of water and aeration for 10 minutes, use 80 mesh net to collect the upper layer of fertilized active eggs, filter the debris with 20 mesh filter screen, and then move to the incubation pool with a density of about 500,000 eggs / m³. The incubation water temperature is 24°C, the salinity is 26-28‰, the pH value is 7.9-8.0, and the aeration method is used for incubation.

[0035] Example 4: Larvae cultivation After 32-36 hours at a water temperature of 24°C, the larvae hatched and broke the membrane. The body length of the newly hatched larvae was about 2.8-3 mm. On the third day after hatching, the larvae were fed with rotifers that were nutritionally enhanced with chlorella. The feeding density was 10 / mL, and the remaining rotifers in the cultivation pool were counted before the second feeding each day, and then the rotifers were supplemented. Artemia nauplii were fed at 8 days old, with a feeding amount of about 100 per fish per day, and the feeding was divided into two times according to the feeding situation. After 11 days, the larvae were fed with artemia nauplii and micro-particle compound feed, twice a day; after 15 days, the feeding amount of artemia nauplii was reduced; and after 20 days, only micro-particle compound feed was fed, three times a day.

[0036] Experimental example: Larvae culture test 5000 larvae (average total length 5.2±0.3 cm, average weight 3.1±0.3 g) were randomly selected from the cultivated larvae on June 17 and transferred to a general net cage culture base (Changzhi Island Culture Base of Zhejiang Ocean University) to carry out net cage culture test, focusing on monitoring survival rate, growth rate and disease occurrence; 20,000 larvae (cultivated in the same batch) were randomly selected and transferred to a deep water net cage culture base (Daimuzimeng Base in Daoshan), to carry out large-scale culture of adult fish.

[0037] The specific conditions of the above-mentioned autumn breeding of Pseudosciaena crocea and spring breeding test in off-season are shown in Table 1.

[0038] Table 1

Claims

1. A method for regulating off-season reproduction of autumnal reproduction of Pseudosciaena crocea, characterized in that, The method comprises the following steps: 1) Detection of Trypanosoma: Before selecting brood fish, blood is taken from the caudal vein to make blood smears, which are then examined under a microscope after staining to ensure that the brood fish are not infected with Trypanosoma; 2) Selection of brood fish: In December to early January every year, select autumn-reproducing large yellow croakers that are more than 2 years old, have a body weight of ≥550 g for females and ≥450 g for males, and have gonads in phase II, and then transfer the fish to a culture pond for temporary cultivation for one week, and then perform a second detection of Trypanosoma; 3) Cultivation of brood fish: After the temporary cultivation, gradually increase the temperature by 1℃ every 2 days to 23-25℃ and maintain the temperature, and at the same time, start to increase the light time by 1 hour every day from the middle or late January to a total light time of 16-20 hours / day, and control the light intensity at 450-600 lx; feed the brood fish with compound feed having a protein content of ≥45% and a fat content of 8-12% and add 5%-8% of chlorella, and feed the brood fish with feed added with a compound vitamin and a compound mineral every 10 days; 4) Artificial reproduction: after the cultivation of the brood fish, monitor the development of the gonads, and when the oocytes of the female fish develop to the end of phase IV and the sperm of the male fish mature, inject the female fish with luteinizing hormone-releasing hormone analogue LRH-A3 to induce spawning at a dose of 2-5 μg / kg, and inject the male fish with the luteinizing hormone-releasing hormone analogue LRH-A3 at a dose of half of that for the female fish, and then place the female and male fish in an egg laying pond at a female to male ratio of 1:1-1:2 after a single or two injections; 5) Artificial incubation: collect the fertilized eggs and place the eggs in an incubation pond, and incubate the eggs under the conditions of a water temperature of 23-25℃, a salinity of 21-30‰, and a pH of 7.85-8.35, and use the air charging method or the flow water method to incubate the eggs, and remove dead eggs at regular time intervals; 6) Cultivation of fry: place the hatched fish in a fry cultivation pond, and feed the fry with rotifers, artemia nauplii, and micro-particle compound feed in sequence after the fry open their mouths, feed the fry with compound feed alone after 20 days of age, and control the environmental noise and disinfect the cultivation pond during the cultivation period. The culture pond in step 2) is an indoor cement pond, a circular glass steel tank, or a PP circular breeding barrel with a diameter of more than 3 m and a water depth of 1.5-2 m, and is provided with a temperature control system, a water inlet and outlet system, an oxygenation device, and a light adjustment device.

2. The method for regulating the off-season reproduction of the autumnal reproduction of Pseudosciaena crocea according to claim 1, characterized in that, The water quality conditions during the cultivation of the brood fish in step 3) are as follows: a dissolved oxygen of ≥6 mg / L, a salinity of 21-30‰, and a pH of 7.85-8.35, and the water quality is detected once a week.

3. The method for regulating the off-season reproduction of large yellow croaker in autumn as described in claim 1, characterized in that, In step 4), the first injection amount of the luteinizing hormone-releasing hormone analogue is 1 / 2 of the total dose, and the remaining amount is injected after 24-48 hours, the egg laying pond is kept in a weak light or dark environment, and the circulating water flow rate is increased.

4. The method for regulating the off-season reproduction of large yellow croaker in autumn as described in claim 1, characterized in that, In step 6), the fry are fed with chlorella-strengthened Brachionus plicatilis at a density of 10-15 pieces / mL after the fry open their mouths, the fry are fed with artemia nauplii at 100-200 pieces per tail per day after 8 days of age, the fry are fed with micro-particle feed after 11 days of age, and the fry are fed with micro-particle feed alone after 20 days of age, and the fry are fed 3-4 times a day.

5. The method for regulating the off-season reproduction of large yellow croaker in autumn as described in claim 1, characterized in that, The culture pond, the incubation pond, and the fry cultivation pond are disinfected with a 15-20 ppm potassium permanganate solution before use, and the brood fish and the fry are prevented from being disturbed by noise of 100-600 Hz during the cultivation period.

6. The method for regulating the off-season reproduction of large yellow croaker in autumn as described in claim 1, characterized in that, ​

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