Oral oxytocic hormone feed for parent fish of large yellow croaker of wild sal family as well as preparation method and application of oral oxytocic hormone feed

Abstract

The invention provides oral oxytocic hormone feed for parent fish of wild sal-family pseudosciaena crocea as well as a preparation method and application of the oral oxytocic hormone feed, and belongs to the technical field of breeding of the wild sal-family pseudosciaena crocea. According to the oral oxytocin feed for the parent fish of the wild sal-family large yellow croaker, provided by the invention, a high-protein floating compound feed is taken as a carrier, and a gonadotropin releasing hormone analogue GnRH-a, a dopamine receptor antagonist DOM, vitamin E powder, immune polysaccharide for fish and seaweed meal are added. The invention further provides a preparation method and application of the oral oxytocic hormone feed for the parent fish of the wild sal-ammoniac pseudosciaena crocea, and provides an oxytocic, insemination and artificial propagation method for the wild sal-ammoniac pseudosciaena crocea, so that the propagation success rate and the fry quality of the wild sal-ammoniac pseudosciaena crocea are remarkably improved; the technical bottlenecks of stress sensitivity, asynchronous oviposition, unstable egg quality and the like of wild parent fish are overcome, and the method has remarkable popularization value and industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology for wild large yellow croaker (Cyprinus naanensis), specifically relating to an oral spawning-inducing hormone feed for broodstock of wild large yellow croaker, its preparation method, and its application. Background Technology

[0002] Naozhou large yellow croaker ( Larimichthys crocea This species, *Pterocarya stenoptera*, is a unique geographical population distributed in the waters around Naozhou Island and adjacent deep-water areas, possessing unique germplasm resources and high economic value. Individuals in this population are generally plump, with tender muscle fibers and a rich flavor, making it an important species for high-end mariculture and fishing. However, due to long-term fisheries development and habitat changes, wild populations have declined significantly, and natural breeding populations are decreasing, making artificial breeding and germplasm conservation efforts urgently needed.

[0003] Currently, China has accumulated relatively mature artificial breeding experience for other geographical groups of large yellow croaker, such as the Fujian-Eastern Guangdong and Daiqu groups, including technical systems for gonad maturation induction, artificial spawning induction, artificial insemination, and seedling cultivation. However, the direct application of these techniques to the Naozhou group of large yellow croaker has significant limitations, especially in the domestication and spawning induction of wild broodstock, mainly manifested in the following prominent problems: 1. Strong stress response and high difficulty in domestication. Wild Naozhou group of large yellow croaker has long inhabited deep-sea areas with high current speed and stable salinity. They are sensitive and vigorous, and extremely sensitive to environmental changes and mechanical operations. During the fishing, transportation, and temporary holding processes, especially during artificial operations (such as injection to induce spawning), they are prone to violent escape, collisions with net walls, abrasions on the body surface, and rapid breathing, which in turn leads to reduced feeding, decreased immunity, and even acute death. This high stress characteristic significantly reduces the availability of broodstock and also restricts the stability of mass spawning induction. 2. Asynchronous gonad development and low concentration of spawning. The gonadal development of wild broodstock is primarily regulated seasonally by various factors, including natural seawater temperature, photoperiod, and food composition. In artificial environments, these environmental factors often cannot be fully simulated, leading to uneven gonadal development and inconsistent maturation processes between males and females, resulting in dispersed spawning times and irregular batches. This asynchrony directly affects the sperm-egg matching efficiency of artificial insemination, thus reducing fertilization and hatching rates. 3. Existing spawning induction methods have significant limitations. Currently, artificial spawning induction commonly uses hormone injections such as GnRH-a and HCG. While effective in cultured broodstock, these methods exhibit poor adaptability in wild broodstock. The injection process requires individual catching, fixation, and injection, increasing both time and labor costs. Furthermore, prolonged out-of-water exposure and strong restraint can trigger severe stress responses. In practice, delayed spawning, decreased egg quality, incomplete spawning, and even spawning failure are common. Moreover, injection-induced spawning requires specialized technicians and standardized operating procedures, demanding high levels of on-site management, which hinders large-scale promotion in fisheries resource protection and restoration. 4. The contradiction between industry demand and technological bottlenecks is prominent. With the development of the aquaculture industry into deep-sea areas and the increased national efforts to protect marine fishery germplasm resources, the demand for artificial breeding and seedling supply of *Large Yellow Croaker* (a type of croaker) continues to rise. However, the lack of low-stress spawning-inducing techniques suitable for wild broodstock limits the large-scale and sustainable development of artificial breeding. Particularly in areas such as resource recovery releases, conservation and purification, and improved seed propagation, higher demands are placed on the safety, stability, and large-scale production capacity of broodstock spawning-inducing techniques. Therefore, there is an urgent need to develop a low-stress, easy-to-operate spawning-inducing method adapted to the physiological characteristics of wild broodstock to significantly improve reproductive success rates and germplasm utilization efficiency, providing reliable technical support for germplasm resource protection and industrial utilization. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the first objective of the present invention is to provide an oral spawning-inducing hormone feed for wild Naozhou large yellow croaker broodstock that has both spawning-inducing and nutritional effects.

[0005] A second objective of this invention is to provide a method for preparing the above-mentioned feed containing oral oxytocin for broodstock of wild large yellow croaker.

[0006] A third objective of this invention is to provide the application of the above-mentioned oral oxytocin feed for wild Naozhou large yellow croaker broodstock or the above-mentioned preparation method in the artificial breeding of wild Naozhou large yellow croaker.

[0007] A fourth objective of this invention is to provide a method for inducing spawning in wild large yellow croaker broodstock.

[0008] A fifth objective of this invention is to provide a method for artificial insemination of wild large yellow croaker from the Naozhou group.

[0009] A sixth objective of this invention is to provide a method for the artificial breeding of wild large yellow croaker from the Naozhou group.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an oral spawning-inducing hormone feed for wild large yellow croaker broodstock that combines spawning-inducing and nutritional effects. Using a high-protein floating compound feed as a carrier, the feed contains, by weight, 0.5g / 100kg~1g / 100kg of gonadotropin-releasing hormone analog GnRH-a, 5g / 100kg~10g / 100kg of dopamine receptor antagonist DOM, 50g / 100kg~100g / 100kg of vitamin E powder, 100g / 100kg~200g / 100kg of fish immunopolysaccharide, and 1kg / 100kg of seaweed powder.

[0011] Preferably, the particle size of the oral oxytocin feed for wild Naozhou large yellow croaker broodstock is 5-8 mm.

[0012] The present invention also provides a method for preparing the above-mentioned oral oxytocin feed for broodstock of wild Naozhou large yellow croaker, comprising the following steps: dissolving GnRH-a and DOM in 0.1% glacial acetic acid solution and spraying it onto the surface of high-protein floating compound feed; after spraying, adding vitamin E powder, fish immunopolysaccharide and seaweed powder, mixing, and drying to obtain the oral oxytocin feed for broodstock of wild Naozhou large yellow croaker.

[0013] Preferably, the ratio of the 0.1% glacial acetic acid solution to the high-protein floating compound feed is (3~5) mL:100g; during the spraying process, the high-protein floating compound feed is stirred at a speed of 30~50 r / min and a temperature of 20~25℃; the mixing time is 5~8 min; and the drying temperature is 35~40℃ and the time is 2~3 h.

[0014] The present invention also provides the application of the above-mentioned oral oxytocin feed for wild Naozhou large yellow croaker broodstock or the above-mentioned preparation method in the artificial breeding of wild Naozhou large yellow croaker.

[0015] The present invention also provides a method for inducing spawning in wild Naozhou large yellow croaker broodstock, comprising the following steps: catching wild Naozhou large yellow croaker, temporarily holding the broodstock, and feeding the broodstock with the aforementioned wild Naozhou large yellow croaker broodstock oral spawning hormone feed 7-10 days before the broodstock naturally reproduce; feeding twice a day, with each feeding amount being 0.25-0.5% of the broodstock's body weight; continuous feeding for 3-5 days.

[0016] Preferably, the water temperature for the temporary rearing of the broodstock is 22~24℃, the salinity is 28~30‰, the dissolved oxygen is ≥6.5 mg / L, the water flow velocity is 0.05~0.10 m / s, and the light intensity is 300~800 lx. During the temporary rearing period, the broodstock are fed twice a day with high-protein chilled fish and high-protein floating compound feed, and twice a week with fresh shellfish or crustaceans.

[0017] Preferably, broodstock are caught using fixed nets or purse seines. After capture, they are transferred to a live-water circulation transport vessel or an oxygenated seawater transport tank. The transport water temperature is 21-23℃, salinity is 28-30‰, dissolved oxygen is ≥7.0mg / L, and density is ≤80kg / m³. 3 The transport water contains 10-15 mg / L of vitamin C.

[0018] The present invention also provides an artificial insemination method for wild Naozhou large yellow croaker. The wild Naozhou large yellow croaker parent fish are induced to spawn according to the above-mentioned spawning induction method, and artificial eggs and artificial sperm are collected. The sperm-egg ratio is 1:(100~150), and fertilized eggs are obtained by dry insemination.

[0019] This invention also provides an artificial breeding method for wild large yellow croaker of the Naozhou group. Fertilized eggs are obtained according to the above-mentioned artificial insemination method, transferred to a hatching facility, hatched until the fry stage, and then transferred to a seedling cultivation pond to obtain seedlings.

[0020] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention organically combines oral oxytocin with nutritional fortification and low-stress environment control to form a systematic spawning induction program, which significantly improves the reproductive success rate and seedling quality of wild Naozhou large yellow croaker. It can simultaneously achieve the comprehensive advantages of high spawning synchronization (36-hour window), low stress mortality (0.8%), and high survival rate (81.4%), overcoming the technical bottlenecks of stress sensitivity, asynchronous spawning, and unstable egg quality in wild broodstock. It has significant promotional value and industrial application prospects. Detailed Implementation

[0021] This invention provides an oral spawning-inducing hormone feed for wild large yellow croaker broodstock that combines spawning-inducing and nutritional effects. Using a high-protein floating compound feed as a carrier, the feed contains, by weight, 0.5g / 100kg~1g / 100kg of gonadotropin-releasing hormone analog GnRH-a, 5g / 100kg~10g / 100kg of dopamine receptor antagonist DOM, 50g / 100kg~100g / 100kg of vitamin E powder, 100g / 100kg~200g / 100kg of fish immunopolysaccharide, and 1kg / 100kg of seaweed powder. The gonadotropin-releasing hormone analog GnRH-a is preferably 0.6g / 100kg, 0.7g / 100kg, 0.8g / 100kg, or 0.9g / 100kg; the dopamine receptor antagonist DOM is preferably 6g / 100kg, 7g / 100kg, 8g / 100kg, or 9g / 100kg; the vitamin E powder is preferably 60g / 100kg, 70g / 100kg, 80g / 100kg, or 90g / 100kg; and the fish immunopolysaccharide is preferably 110g / 100kg, 120g / 100kg, 130g / 100kg, 140g / 100kg, 150g / 100kg, 160g / 100kg, 170g / 100kg, 180g / 100kg, or 190g / 100kg.

[0022] In this invention, the fish immunopolysaccharide is preferably a compound immunopolysaccharide preparation with Astragalus membranaceus as the main component, rich in polysaccharides, flavonoids, β-glucan, etc., with a β-glucan mass fraction ≥50%, used to enhance the non-specific immunity and stress resistance of broodstock. The particle size of the orally administered spawning-inducing hormone feed for wild Naozhou large yellow croaker broodstock is 5-8 mm, preferably 6-7 mm. This invention uses a high-protein buoyant compound feed as a carrier and controls the feed particle size to ensure palatability and suspension time, facilitating natural feeding of wild Naozhou large yellow croaker broodstock in the water.

[0023] In this invention's formulation, gonadotropin-releasing hormone analogue (GnRH-a, purity ≥98%) stimulates the activation of the hypothalamus-pituitary-gonadal axis, promoting gonadal maturation; the dopamine receptor antagonist DOM inhibits the inhibitory effect of dopamine on gonadotropin release, and when combined with GnRH-a, significantly increases the secretion levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH); vitamin E powder, as a fat-soluble antioxidant, improves the stability of oocyte membranes and promotes the synthesis of gonadal steroids, thereby enhancing egg quality and sperm motility; fish immunopolysaccharides enhance the immunity of broodstock and reduce stress responses that may occur during spawning induction; and seaweed powder, as a natural attractant, provides trace elements and promotes feeding, ensuring sufficient intake of hormone carrier feed. The uniform mixing of all components satisfies the dual needs of wild broodstock for hormones and nutrition during spawning induction, while maintaining a stable intake of effective components over a 3-5 day feeding cycle.

[0024] This invention also provides a method for preparing the above-mentioned oral spawning-inducing hormone feed for wild Naozhou grouper broodstock, comprising the following steps: GnRH-a and DOM are dissolved in a 0.1% glacial acetic acid solution and sprayed onto the surface of a high-protein floating compound feed; after spraying, vitamin E powder, fish immunopolysaccharide, and seaweed powder are added, mixed, and dried to obtain the oral spawning-inducing hormone feed for wild Naozhou grouper broodstock. The ratio of the 0.1% glacial acetic acid solution to the high-protein floating compound feed is (3~5) mL:100g, preferably 4 mL:100g, to ensure complete dissolution of the hormone in the solution and facilitate uniform spraying; during the spraying process, an atomizing nozzle is used, and the feed is evenly sprayed along the feed stirring shaft at room temperature (20~25℃), with the stirring speed controlled at 30~50 r / min to prevent feed particle breakage caused by mechanical stirring. The mixing time is preferably 5~8 min; the vitamin E powder, immunopolysaccharide, and seaweed powder are fully adhered to the feed surface after mixing. After mixing as described in this invention, the feed is placed in a low-temperature forced-air drying device and dried at 35-40°C for 2-3 hours until the feed moisture content drops below 10% to maintain hormone activity and prevent mold growth. The dried feed should be sealed and stored in a dark, low-humidity (relative humidity < 60%) environment, preferably within 6 hours of preparation, and no longer than 24 hours, to ensure that the active ingredients do not undergo significant degradation, thereby guaranteeing the stability and repeatability of the oxytocin-inducing effect.

[0025] The present invention also provides a method for inducing spawning in wild Naozhou large yellow croaker broodstock, comprising the following steps: catching wild Naozhou large yellow croaker, temporarily holding the broodstock, and feeding the broodstock with the aforementioned wild Naozhou large yellow croaker broodstock oral spawning hormone feed 7-10 days before the broodstock naturally reproduce; feeding twice a day, with each feeding amount being 0.25-0.5% of the broodstock's body weight; continuous feeding for 3-5 days.

[0026] The broodstock harvesting method described in this invention preferably employs fixed nets or purse seines, with the preferred harvesting time being during the early morning when light levels are low. After capture, the fish are immediately transferred to a live-water circulating transport vessel or an oxygen-filled seawater transport tank. The transport water temperature is 21-23°C, salinity is 28-30‰, dissolved oxygen is ≥7.0 mg / L, and density is ≤80 kg / m³. 3 The transport water contains 10-15 mg / L of vitamin C to alleviate stress response. After the broodstock arrive at the indoor factory-produced cement tank, they are preferably acclimatized by gradual water changes over 5-10 minutes to control the temperature and salinity differences within ±1℃ and ±1‰, respectively, to prevent osmotic pressure shock.

[0027] The water temperature for the temporary rearing of broodstock fish in this invention is 22-24℃, the salinity is 28-30‰, the dissolved oxygen is ≥6.5 mg / L, preferably 6.5-7.5 mg / L, the water flow velocity is 0.05-0.10 m / s, and the light intensity is 300-800 lx; ​​the water volume is not less than 30 m³. 3 The preferred holding period is 7-10 days. During this period, the broodstock should be fed twice daily (8:00-9:00 AM and 4:00-5:00 PM) with high-protein chilled fish and high-protein floating formulated feed, with each feeding amount preferably 1% of the broodstock's weight. The high-protein chilled fish and high-protein floating formulated feed are preferably mixed in a 3:7 ratio, with the high-protein chilled fish preferably including mackerel. The high-protein floating formulated feed is the carrier feed in the broodstock's oral spawning hormone feed. Fresh shellfish or crustaceans, preferably including oysters, should be supplemented 1-2 times per week as reproductive nutrition fortification feed, with each supplemental feeding amount preferably 1-3% of the broodstock's weight.

[0028] During the temporary holding period, the present invention preferably minimizes human interference, avoids frequent fishing or mechanical operations, and observes the feeding situation, swimming status and body surface integrity daily. Any abnormal individuals should be isolated in time to ensure that the parent fish entering the spawning stage are healthy, have normal gonad development and low stress levels.

[0029] This invention involves feeding the broodstock with prepared spawning-inducing feed 7-10 days before the natural breeding season (January-February) of the large yellow croaker (Croatia acutissima). Feeding is done twice daily (8:00-9:00 AM and 4:00-5:00 PM), preferably at 0.25%-0.5% of the broodstock's body weight each time, resulting in a total daily feed of 0.5%-1.0% of the broodstock's body weight. This feeding continues for 3-5 days to ensure the broodstock continuously receive an effective dose of hormones to achieve synchronized gonadal maturation. After feeding ceases, the broodstock's behavior is closely monitored for the next 24-48 hours, focusing on signs of spawning such as frequent leaping out of the water, schooling, darkening of body color, abdominal swelling, and redness and swelling of the vent. Artificial egg collection and insemination are promptly performed upon detection of these signs to ensure the acquisition of mature, high-quality eggs and sperm.

[0030] This invention also provides an artificial insemination method for wild *Large Yellow Croaker* (a type of culter fish). The method involves inducing spawning in broodstock of wild *Large Yellow Croaker* using the aforementioned spawning-inducing method, followed by artificial egg and sperm collection. The sperm-egg ratio is 1:1 (100-150), preferably 1:110, 1:120, 1:130, or 1:140. Dry insemination is used to obtain fertilized eggs. As an optional implementation, after the broodstock exhibit signs of spawning and sperm release, artificial egg and sperm collection is performed immediately. A soft polyethylene container is used to collect the eggs to avoid mechanical damage to the egg membrane. Semen collected from a single ejaculation by a mature male is selected, kept dry, and protected from direct sunlight to maintain sperm motility. Dry insemination is used. After mixing the sperm and eggs in the specified ratio, 5-8 times the volume of clean seawater (salinity 28‰) is immediately added for activation. The mixture is gently stirred for 5-10 seconds to promote fertilization. The activation time is ≤1 minute, preferably 30 seconds.

[0031] This invention also provides an artificial breeding method for wild large yellow croaker (Cyprinus naanensis). Fertilized eggs are obtained using the artificial insemination method described above, transferred to an incubation facility, and incubated until hatching. After hatching, the fry are transferred to a seedling rearing pond to obtain seedlings. As an optional implementation, this invention rapidly transfers the fertilized eggs to the incubation facility, controlling the incubation water temperature at 23-25℃, maintaining a salinity of 28‰, dissolved oxygen at 6.5-7.5 mg / L, pH at 7.8-8.2, and light intensity at 300-500 lx. A micro-flow environment of 0.02-0.05 m / s is maintained to prevent egg adhesion and sedimentation. During incubation, a microporous aeration device should be installed to maintain slow water circulation and prevent air bubbles from adhering to the surface of the embryos. Inspections should be conducted 2-3 times daily, and dead eggs should be removed promptly. In this invention, the incubation period from fertilized eggs to hatching generally takes 36-48 hours. After hatching, the fry are immediately transferred to a seedling rearing pond, and rotifers are fed for the first time according to the mouth-opening time of the fry. The method described in this invention can effectively ensure that the hatching rate of fertilized eggs remains stable at over 85% and improve the survival rate of seedlings.

[0032] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] In a specific embodiment of the present invention, the fish immune polysaccharide was purchased from Henan Huachu Biotechnology Co., Ltd., product name: immune polysaccharide, item number: X2079190.

[0034] Unless otherwise specified, the following embodiments are all conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Example 1 A spawning-inducing hormone-inducing feed for wild-caught large yellow croaker broodstock, possessing both spawning-inducing and nutritional effects, is formulated using a high-protein floating compound feed as a carrier. By carrier weight, it contains 0.5 g / 100 kg of a gonadotropin-releasing hormone analog GnRH-a, 5 g / 100 kg of a dopamine receptor antagonist DOM, 50 g / 100 kg of vitamin E powder, 100 g / 100 kg of fish immunopolysaccharide, and 1 kg / 100 kg of seaweed powder. The particle size of this spawning-inducing hormone-inducing feed for wild-caught large yellow croaker broodstock is 5 mm.

[0037] Preparation method: GnRH-a and DOM are dissolved in a 0.1% glacial acetic acid solution. The ratio of the 0.1% glacial acetic acid solution to the high-protein floating compound feed is 3 mL: 100 g. Using an atomizing nozzle, the solution is evenly sprayed along the feed stirring shaft at room temperature (20℃) with the stirring speed controlled at 30 r / min. The 0.1% glacial acetic acid solution containing GnRH-a and DOM is then sprayed onto the surface of the high-protein floating compound feed. Subsequently, vitamin E powder, fish immunopolysaccharides, and seaweed powder are added and mixed for 5 minutes to ensure they fully adhere to the feed surface. After mixing, the feed is placed in a low-temperature forced-air drying device and dried at 35℃ for 3 hours until the feed moisture content drops below 10%.

[0038] Example 2 A spawning-inducing hormone-inducing feed for wild-caught large yellow croaker broodstock, possessing both spawning-inducing and nutritional effects, is formulated using a high-protein floating compound feed as a carrier. By carrier weight, it contains 1 g / 100 kg of gonadotropin-releasing hormone analog GnRH-a, 10 g / 100 kg of dopamine receptor antagonist DOM, 100 g / 100 kg of vitamin E powder, 200 g / 100 kg of fish immunopolysaccharide, and 1 kg / 100 kg of seaweed powder. The particle size of this spawning-inducing hormone-inducing feed for wild-caught large yellow croaker broodstock is 8 mm.

[0039] Preparation method: GnRH-a and DOM are dissolved in a 0.1% glacial acetic acid solution. The ratio of the 0.1% glacial acetic acid solution to the high-protein floating compound feed is 5 mL: 100 g. Using an atomizing nozzle, the solution is evenly sprayed along the feed stirring shaft at room temperature (25℃) with the stirring speed controlled at 50 r / min. The 0.1% glacial acetic acid solution containing GnRH-a and DOM is then sprayed onto the surface of the high-protein floating compound feed. Subsequently, vitamin E powder, fish immunopolysaccharides, and seaweed powder are added and mixed for 8 minutes to ensure full adhesion to the feed surface. After mixing, the feed is placed in a low-temperature forced-air drying device and dried at 40℃ for 2 hours until the feed moisture content drops below 10%.

[0040] Example 3 A spawning-inducing hormone-inducing feed for wild-caught large yellow croaker broodstock, possessing both spawning-inducing and nutritional effects, is formulated using a high-protein floating compound feed as a carrier. By carrier weight, it contains 0.8 g / 100 kg of a gonadotropin-releasing hormone analog (GnRH-a), 8 g / 100 kg of a dopamine receptor antagonist (DOM), 80 g / 100 kg of vitamin E powder, 150 g / 100 kg of fish immunopolysaccharides, and 1 kg / 100 kg of seaweed powder. The particle size of this spawning-inducing hormone-inducing feed for wild-caught large yellow croaker broodstock is 6 mm, and the crude protein content is ≥48%.

[0041] Preparation method: GnRH-a and DOM are dissolved in a 0.1% glacial acetic acid solution. The ratio of the 0.1% glacial acetic acid solution to the high-protein floating compound feed is 4 mL: 100 g. Using an atomizing nozzle, the solution is evenly sprayed along the feed stirring shaft at room temperature (22℃) with the stirring speed controlled at 40 r / min. The 0.1% glacial acetic acid solution containing GnRH-a and DOM is sprayed onto the surface of the high-protein floating compound feed. Subsequently, vitamin E powder, fish immunopolysaccharide, and seaweed powder are added and mixed for 6 minutes to ensure full adhesion to the feed surface. After mixing, the feed is placed in a low-temperature forced-air drying device and dried at 38℃ for 2.5 hours until the feed moisture content drops below 10%.

[0042] Comparative Example 1 A labor-inducing feed, which differs from Example 3 only in that it does not contain vitamin E powder, immunopolysaccharides, and seaweed powder.

[0043] Using a high-protein floating compound feed as a carrier, 0.8 g / 100 kg of gonadotropin-releasing hormone analog GnRH-a and 8 g / 100 kg of dopamine receptor antagonist DOM were added by weight of the carrier. The feed particle size was 6 mm.

[0044] Preparation method: GnRH-a and DOM were dissolved in a 0.1% glacial acetic acid solution. The ratio of the 0.1% glacial acetic acid solution to the high-protein floating compound feed was 4 mL:100 g. Using an atomizing nozzle, the solution was evenly sprayed along the feed stirring shaft at room temperature (22℃) with the stirring speed controlled at 40 r / min. The 0.1% glacial acetic acid solution containing GnRH-a and DOM was then sprayed onto the surface of the high-protein floating compound feed. Subsequently, the feed was placed in a low-temperature forced-air drying device and dried at 38℃ for 2.5 h until the feed moisture content dropped below 10%.

[0045] Example 4 A method for inducing spawning in wild Naozhou large yellow croaker parent fish: Harvesting wild Naozhou grouper (large yellow croaker): Broodstock are caught using fixed nets during the early morning low-light period. After capture, they are immediately transferred to a live-water circulating transport vessel. The transport water temperature is 21℃, salinity 28‰, dissolved oxygen ≥7.0mg / L, and density ≤80kg / m³. 3 The transport water contains 10 mg / L of vitamin C to alleviate stress response. After the broodstock arrive at the indoor factory-style cement tank, they undergo a 5-minute gradual water change acclimatization process to control the temperature and salinity differences within ±1℃ and ±1‰, respectively, to prevent osmotic pressure shock.

[0046] Broodstock Temporary Holding: The water temperature for broodstock holding should be 22℃, salinity 28‰, dissolved oxygen ≥6.5 mg / L, water flow velocity 0.05m / s, and light intensity 500lx; the water volume should be no less than 30 m³. 3 The temporary holding period is 7 days. During this period, the broodstock will be fed twice daily (8:00 AM and 4:00 PM) with a mixture of high-protein chilled fish and high-protein floating formulated feed in a 3:7 ratio. The high-protein chilled fish will be mackerel. Each feeding will be 0.25% of the broodstock's body weight. Fresh shellfish (oysters) will be supplemented twice weekly as a reproductive nutrition-enhancing feed, at a rate of 2% of the broodstock's body weight each time.

[0047] Induced spawning: Seven days before the natural breeding season of the large yellow croaker (January to February), the spawning-inducing feed described in Example 3 was started in the broodstock holding tank. Feeding was conducted twice daily (8:00-9:00 AM and 4:00-5:00 PM), with each feeding amount being 0.25% of the broodstock's body weight. Feeding continued for five days, then stopped. During the following 24-48 hours, close monitoring of the broodstock's behavior was conducted, focusing on signs of spawning such as frequent leaping out of the water, group swimming, deepening body color, abdominal swelling, and redness and swelling of the vent. Artificial egg collection and insemination were promptly performed upon detection of these signs to ensure the acquisition of mature, high-quality eggs and sperm.

[0048] Example 5 A method for inducing spawning in wild Naozhou large yellow croaker parent fish: Harvesting wild Naozhou grouper (large yellow croaker): Broodstock are caught using purse seine netting during the early morning hours when light is low. After capture, the broodstock are immediately transferred to oxygenated seawater transport tanks. The transport water temperature is 23℃, salinity 30‰, dissolved oxygen ≥7.0mg / L, and density ≤80kg / m³. 3 The transport water contains 15 mg / L of vitamin C to alleviate stress response. After the broodstock arrive at the indoor factory-style cement tank, they undergo a 10-minute gradual water change acclimatization process to control the temperature and salinity differences within ±1℃ and ±1‰, respectively, to prevent osmotic pressure shock.

[0049] Broodstock holding: The water temperature for broodstock holding should be 24℃, salinity 30‰, dissolved oxygen ≥6.5 mg / L, water flow velocity 0.10 m / s, and light intensity 800 lx; ​​the water volume should be no less than 30 m³. 3 The temporary holding period is 10 days. During this period, the broodstock will be fed twice daily (8:00 AM and 4:00 PM) with a mixture of high-protein chilled fish and high-protein floating formulated feed in a 3:7 ratio. The high-protein chilled fish will be mackerel. Each feeding will be 1% of the broodstock's body weight. Fresh crustaceans (oysters) will be supplemented twice weekly as a reproductive nutrition-enhancing feed, with each feeding being 2% of the broodstock's body weight.

[0050] 7-10 days before the parent fish reproduce naturally, feed the wild Naozhou large yellow croaker parent fish with oral spawning hormone feed; feed twice a day, each time the amount is 0.25-0.5% of the parent fish's body weight; feed continuously for 3-5 days.

[0051] Induced spawning: Ten days before the natural breeding season of the large yellow croaker (January to February), the spawning-inducing feed described in Example 3 was started in the broodstock holding tank. Feeding was done twice daily (8:00-9:00 AM and 4:00-5:00 PM), with each feeding amount being 0.5% of the broodstock's body weight. Feeding continued for five days, then feeding was stopped. During the following 24-48 hours, close monitoring of the broodstock's behavior was conducted, focusing on signs of spawning such as frequent jumping out of the water, schooling, darkening of body color, abdominal swelling, and redness and swelling of the vent. Artificial egg collection and insemination were promptly performed upon detection of these signs to ensure the acquisition of mature, high-quality eggs and sperm.

[0052] Example 6 A method for artificial insemination of wild *Large Yellow Croaker* from Naozhou: Following the method in Example 4, spawning was induced in broodstock of wild *Large Yellow Croaker*. Once the broodstock showed signs of ovulation and sperm release, artificial egg and sperm collection was immediately performed. A soft polyethylene container was used to collect the eggs to avoid mechanical damage to the egg membrane. Sperm collected from a single ejaculation of a mature male was carefully selected, kept dry, and protected from direct sunlight to maintain sperm motility. Dry insemination was used. The sperm and eggs were mixed at a ratio of 1:150, and immediately 5 times the volume of clean seawater (salinity 28‰) was added for activation. The mixture was gently stirred for 10 seconds to promote fertilization, and the activation time was 1 minute.

[0053] Example 7 A method for artificial insemination of wild *Large Yellow Croaker* (a type of culter fish) is disclosed. Following the method described in Example 5, spawning is induced in broodstock of wild *Large Yellow Croaker*. Once the broodstock exhibit signs of ovulation and sperm release, artificial egg and sperm collection is immediately performed. During egg collection, a soft polyethylene container is used to collect the eggs to avoid mechanical damage to the egg membrane. Sperm collected is selected from the single ejaculation of mature male fish, kept dry, and protected from direct sunlight to maintain sperm motility. Dry insemination is employed. The sperm and eggs are mixed at a ratio of 1:100, and immediately 8 times the volume of clean seawater (salinity 28‰) is added for activation. Gently stirring for 5 seconds promotes fertilization, and the activation time is 30 seconds.

[0054] Example 8 An artificial breeding method for wild *Cyprinus naanus*: Fertilized eggs were obtained according to the method in Example 6. The fertilized eggs were then rapidly transferred to an incubation facility. The incubation water temperature was controlled at 23℃, the salinity at 28‰, dissolved oxygen at 6.5 mg / L, pH at 7.8, and light intensity at 300 lx. A micro-flow environment of 0.02 m / s was maintained to prevent egg adhesion and sedimentation. During incubation, a microporous aeration device was installed to maintain slow water circulation and prevent air bubbles from adhering to the surface of the embryos. The facility was inspected twice daily, and dead eggs were removed promptly. After hatching, the fertilized eggs were immediately transferred to a fry rearing pond, and rotifers were fed for the first time according to the time of mouth opening in the fry.

[0055] Example 9 A method for artificial breeding of wild *Cyprinus naanensis*: Fertilized eggs were obtained according to the method in Example 7. The fertilized eggs were then rapidly transferred to an incubation facility. The incubation water temperature was controlled at 25℃, the salinity at 28‰, dissolved oxygen at 7.5 mg / L, pH at 8.2, and light intensity at 500 lx. A micro-flow environment of 0.05 m / s was maintained to prevent egg adhesion and sedimentation. During incubation, a microporous aeration device was installed to maintain slow water circulation and prevent air bubbles from adhering to the surface of the embryos. Inspections were conducted three times daily, and dead eggs were removed promptly. After hatching, the fertilized eggs were immediately transferred to a fry rearing pond, and rotifers were fed for the first time according to the time of mouth opening in the fry.

[0056] Experimental Example 1 Comparison of the effects of different spawning induction methods on wild Naozhou large yellow croaker: 1. Source and basic information of broodstock Fishing time and sea area: Mid-February 2025, fishing will be carried out in the waters east of Naozhou Island, Zhanjiang City, Guangdong Province, with a water depth of 18-25 m and an ocean current speed of 0.15-0.25 m / s.

[0057] Fishing method: Fixed nets are used, and the time for each net haul-up is controlled within 30 minutes. Fishing operations are carried out during the low light period from 5:30 to 6:30 in the morning to reduce stress on the parent fish.

[0058] Broodstock condition: Female broodstock averaged 25.6 cm in length and 0.7 kg in weight; male broodstock averaged 22.2 cm in length and 0.5 kg in weight. They showed no obvious external damage and were highly vigorous. Gonadal development was in stage IV, with eggs ranging from 0.55 to 0.65 mm in diameter and ovarian weight accounting for 10.2% to 12.1% of body weight.

[0059] 2. Transfer and temporary care Transfer method: Immediately after capture, the vessel is transferred to a circulating freshwater transport hold, where the water temperature is maintained at 22±0.5℃, salinity at 29±0.5‰, and dissolved oxygen at ≥7.0 mg / L. Vitamin C (concentration 12 mg / L) is added to the water in the hold to alleviate transport stress; the transport density is controlled at 75 kg / m³. 3 Within 2 hours, the transportation time shall not exceed 2 hours.

[0060] Temporary holding facilities: Indoor cement pool (4m×4m×2m, volume 32m³) 3 The surrounding area is fitted with shade nets that provide 70% shading to reduce light stimulation.

[0061] Water conditions: During the temporary rearing period, the water temperature was maintained at 23±0.5℃, the salinity at 29±0.5‰, the dissolved oxygen at 6.8~7.2mg / L, the flow rate at 0.08 m / s, and the light intensity at 400±50 lx.

[0062] Feeding and management: The temporary holding period is 8 days. Feed twice a day (8:30 and 16:30). The feed is a mixture of fresh fish (mackerel) and high-protein compound feed in a 3:7 ratio. The total daily feed amount accounts for 1% of the body weight of the broodstock. Fresh shellfish (oysters) are added once a week as a reproductive nutrition fortification, accounting for 2% of the body weight of the broodstock.

[0063] 3. Induction of labor (1) Feeding method for spawning-inducing feed: Feed the spawning-inducing feed prepared in Example 3; the feeding cycle is 4 consecutive days, twice a day (8:30 and 16:30), the amount of feed given at one time is 0.4% of the body weight of the parent fish, and the total amount given each day is 0.8%; the feeding rate of each batch of parent fish is ≥95% when feeding, and the uneaten part is removed within 10 minutes. Keep the water body stable and move slowly during the feeding process to prevent the parent fish from being startled.

[0064] (2) Traditional injection-induced spawning method: GnRH-a 10 μg / kg body weight + DOM 5 mg / kg body weight. Injection method: On the day of spawning (9:00 am), catch the parent fish one by one using soft nets, and control the time out of the water for each time to <1 min; place the parent fish on a wet towel and inject them into the dorsal muscle. The injection needle is a disposable fish syringe (needle length 12 mm, needle diameter 0.6 mm); the drug solution is dissolved in 0.9% physiological saline solution, the injection volume is 0.5-0.8 mL per fish, and the injection angle is about 45°. After the injection, immediately return them to the cement pond, observe their swimming status and continue to oxygenate.

[0065] 4. Ovulation monitoring and artificial insemination (1) Feeding method for spawning: About 36 hours after stopping feeding, start observing whether the parent fish show signs of spawning such as frequent grouping, accelerated swimming, obvious abdominal swelling, and congestion of the vent.

[0066] (2) Traditional injection spawning method: Some female fish begin to show signs of spawning 24 to 30 hours after injection, but the spawning time is scattered, and some female fish do not start to spawn until 48 hours after injection.

[0067] Female fish exhibiting signs of fertilization were immediately harvested for egg collection, using soft polyethylene containers. Male semen collection was conducted entirely in a dry environment, using a 1 mL disposable syringe, and stored away from light; it was to be used within 5 minutes. Dry insemination was performed using a sperm-to-egg ratio of 1:120, adding 5 times the volume of clean seawater with a salinity of 28‰ and a temperature of 24℃. The mixture was gently stirred for 8 seconds and then allowed to stand for 30 seconds to complete activation.

[0068] 5. Hatching and Seedling Cultivation The incubation facility consists of circular fiberglass incubation tanks (1.5 m in diameter, 1.0 m in water depth, and 1.8 m³ in volume). 3The water temperature was 24±0.5℃, the salinity was 28±0.5‰, the dissolved oxygen was 6.8~7.5 mg / L, the pH value was 7.9~8.1, the light intensity was 400±30 lx, and the flow rate was 0.03 m / s.

[0069] Inspect three times daily, removing dead eggs and replenishing with fresh water. The incubation period is approximately 38 hours. Feed rotifers (density 5-8 ind / mL) starting 48 hours after hatching, and gradually introduce copepods or artichokes starting on day 4. Gradually introduce formulated feed starting on day 20. Results from different spawning induction methods are shown in the table below: Table 1. Statistical results of various indicators for different labor induction methods

[0070] Compared with the feeding method of spawning-inducing feed (the method of this invention), the traditional injection spawning-inducing method for wild large yellow croaker from the Naozhou group resulted in a decrease of approximately 12.1%, 11.2%, and 10.1% in core reproductive indicators such as fertilization rate, hatching rate, and fry emergence rate, respectively. Furthermore, the mortality rate due to spawning stress increased significantly, from 0.8% to 5.2%, and the fry deformity rate also increased from 1.2% to 3.8%. The fry survival rate on day 30 decreased by approximately 12.7 percentage points. Although traditional injection spawning induction is still used in indoor broodstock culture, its high stress characteristics and asynchronous spawning significantly limit reproductive efficiency and fry quality in the artificial breeding of wild large yellow croaker from the Naozhou group. The oral spawning-inducing method of this invention has significant advantages in reducing stress, improving spawning synchronicity, and optimizing egg quality, making it particularly suitable for large-scale domestication and mass breeding of wild broodstock.

[0071] Experimental Example 2 Investigating the effects of nutritional fortification and environmental control on wild large yellow croaker from Naozhou: 1. Source and basic information of broodstock Fishing time and sea area: Mid-February 2025, fishing will be carried out in the waters east of Naozhou Island, Zhanjiang City, Guangdong Province, with a water depth of 18-25 m and an ocean current speed of 0.15-0.25 m / s.

[0072] Fishing method (low light operation not strictly controlled): Fixed net operation is used, with a single net retrieval time of about 35 minutes. Fishing operation time is from 16:30 to 18:30 in the afternoon.

[0073] Parent fish body condition: Females average body length 25.8cm and weight 0.7kg; males average body length 23.2cm and weight 0.4kg; gonadal development stage IV, egg diameter 0.54~0.64 mm, ovary weight as a percentage of body weight 10.0~11.8%.

[0074] 2. Transfer and temporary care Transport method: Same as in Experiment 1.

[0075] Temporary care facilities: Same as in Experiment 1.

[0076] Water conditions (unstable): water temperature fluctuates between 22 and 25°C, salinity is 27 to 30‰, dissolved oxygen is as low as 5.8 mg / L, and flow velocity is unstable (0.03 to 0.12 m / s).

[0077] Feeding and management: The temporary holding period is 6 days. Feeding is done twice a day (8:30 and 16:30). The feed is a mixture of fresh fish (mackerel) and high-protein compound feed in a 5:5 ratio. The total daily feed amount accounts for 1% of the body weight of the parent fish. No additional reproductive nutrition-enhancing feed such as shellfish is provided.

[0078] 3. Induction of labor Simple oral induction of labor method: Feed the feed of Comparative Example 1 in the same way as the feeding method of the labor-inducing feed in Experimental Example 1 and Example 3.

[0079] 4. Egg laying and fertilization About 40 hours after feeding was stopped, some female fish began to lay eggs, but the spawning synchronization was poor, and the spawning rate was about 65%.

[0080] Female fish exhibiting signs of fertilization should be immediately captured and their eggs collected using a soft polyethylene container. Male semen should be collected in a completely dry environment using a 1 mL disposable syringe, stored away from light, and used within 5 minutes. Dry insemination: A sperm-to-egg ratio of 1:120 is used, with 5 times the volume of eggs added to clean seawater at a salinity of 28‰ and a temperature of 24℃. The mixture is gently stirred for 8 seconds and then allowed to stand for 30 seconds to complete activation.

[0081] 5. Hatching and Seedling Cultivation The incubation conditions were the same as in Experiment 1.

[0082] The incubation period was approximately 39 hours. Some fertilized eggs underdeveloped, and the proportion of dead eggs was relatively high. The results are shown in the table below: Table 2. Effects of nutritional fortification and environmental control on various indicators

[0083] Compared to the method of feeding oxytocin-induced feed (the method of this invention), the method of simply administering oral oxytocin, although also using oral oxytocin, suffers from reduced egg-laying synchronicity (from over 92% to 65%) due to the lack of nutritional fortification components such as vitamin E, immunopolysaccharides, and seaweed powder, as well as large fluctuations in environmental conditions such as water temperature, dissolved oxygen, and flow rate. This results in a 5-12 percentage point decrease in fertilization rate, hatching rate, and survival rate, and an increase in stress-induced mortality. This indicates that the method of this invention not only relies on oral oxytocin but also forms a systematic technical solution through nutritional fortification and environmental control, which is key to improving reproductive efficiency.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A spawning-inducing hormone feed for wild *Large Yellow Croaker* broodstock that combines spawning-inducing and nutritional effects, characterized in that... Using high-protein floating compound feed as a carrier, add 0.5g / 100kg~1g / 100kg of gonadotropin-releasing hormone analog GnRH-a, 5g / 100kg~10g / 100kg of dopamine receptor antagonist DOM, 50g / 100kg~100g / 100kg of vitamin E powder, 100g / 100kg~200g / 100kg of fish immunopolysaccharide and 1kg / 100kg of seaweed powder by weight.

2. The feed for broodstock of wild large yellow croaker (Cyprinus naan) according to claim 1, characterized in that, The particle size of the feed containing oxytocin orally administered spawning hormones to the parent fish of wild Naozhou large yellow croaker is 5-8 mm.

3. The method for preparing the spawning-inducing hormone feed for wild large yellow croaker broodstock according to any one of claims 1 to 2, characterized in that, Includes the following steps: GnRH-a and DOM were dissolved in a 0.1% glacial acetic acid solution and sprayed onto the surface of a high-protein floating compound feed. After spraying, vitamin E powder, fish immunopolysaccharide, and seaweed powder were added, mixed, and dried to obtain an oral spawning hormone feed for wild Naozhou large yellow croaker broodstock.

4. The preparation method according to claim 3, characterized in that, The ratio of the 0.1% glacial acetic acid solution to the high-protein floating compound feed is (3~5) mL:100g; during the spraying process, the high-protein floating compound feed is stirred at a speed of 30~50 r / min and a temperature of 20~25℃; the mixing time is 5~8 min; the drying temperature is 35~40℃ and the time is 2~3 h.

5. The application of the oral spawning hormone feed for wild Naozhou large yellow croaker broodstock as described in any one of claims 1 to 2, or the preparation method as described in any one of claims 3 to 4, in the artificial breeding of wild Naozhou large yellow croaker.

6. A method for inducing spawning in wild large yellow croaker broodstock, characterized in that, Includes the following steps: Wild large yellow croaker from Naozhou is caught and temporarily held as parent fish. 7-10 days before the parent fish reproduce naturally, the parent fish are fed with oral spawning hormone feed as described in any one of claims 1-2. The feed is given twice a day, with each feeding amount being 0.25-0.5% of the parent fish's body weight. The feeding is continued for 3-5 days.

7. The labor-inducing method according to claim 6, characterized in that, The water temperature for the temporary rearing of the broodstock is 22~24℃, the salinity is 28~30‰, the dissolved oxygen is ≥6.5 mg / L, the water flow velocity is 0.05~0.10 m / s, and the light intensity is 300~800 lx. During the temporary rearing period, the broodstock are fed twice a day with high-protein chilled fish and high-protein floating compound feed, and twice a week with fresh shellfish or crustaceans.

8. The labor-inducing method according to claim 6, characterized in that, Broodstock are caught using fixed nets or purse seines. After capture, they are transferred to live-water circulating transport vessels or oxygenated seawater transport tanks. The transport water temperature is 21-23℃, salinity is 28-30‰, dissolved oxygen is ≥7.0mg / L, and density is ≤80kg / m³. 3 The transport water contains 10-15 mg / L of vitamin C.

9. A method for artificial insemination of wild large yellow croaker (Cyprinus naanensis), characterized in that, The spawning induction method according to any one of claims 6 to 8 is used to induce spawning in wild large yellow croaker broodstock, with artificial egg collection and artificial sperm collection; the sperm-egg ratio is 1:(100-150), and fertilized eggs are obtained by dry insemination.

10. A method for the artificial breeding of wild large yellow croaker (Cyprinus naanensis), characterized in that, The artificial insemination method according to claim 9 obtains fertilized eggs, which are then transferred to an incubation facility for incubation until the seedling stage. After hatching, the eggs are transferred to a seedling cultivation pond to obtain seedlings.

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