Artificial auxiliary natural breeding method for eels

Abstract

The invention belongs to the field of aquaculture, discloses an artificial-assisted natural breeding method for eels, and aims to solve the problems that in the artificial breeding technology of the eels, sexual gland development of male and female fishes is not synchronous, the induced spawning time is difficult to master, and the survival time of newly hatched larva fishes is short. By combining technical means such as habitat simulation, synchronous maturation of sexual glands of parent eels is assisted, natural mating of eels is promoted, and the defects of the artificial breeding technology are overcome. Compared with a disclosed breeding technology, the method has the advantages that the dosage of pituitary gland and chorionic gonadotropin is remarkably reduced, the physiological pressure of the parent fish is reduced, high-quality gametes are obtained and the gonad development synchronization rate of the eel parent fish is increased to 66% from 18% while the ripening effect is ensured by combining a habitat simulation technology through the synergistic effect of hormones; the hatching rate of eel fertilized eggs is increased from 48% to 78%, and the survival time of eel fries is prolonged from 6.8 days to 19.8 days.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture, specifically relating to a method for artificially assisted natural reproduction of eels. Background Technology

[0002] Artificial breeding of eels (including all species of the genus *Anguilla* such as the Japanese eel, European eel, and American eel) has long been a global challenge. Artificially farmed eels cannot mature and spawn naturally, thus necessitating the development of techniques for artificially inducing gonadal maturation and spawning. Currently, only Japan has reported successful fully artificial reproduction of the Japanese eel (Ijiri, S., Tsukamoto, K., Chow, S., Kurogi, H., Adachi, S. & Tanaka, H. 2011. Controlled reproduction in the Japanese eel (Anguilla japonica), past and present. *Aquaculture Europe* 36: 13–17). Currently, scholars from various countries have not been able to achieve ideal results by referring to this technology (Qiu Jixin, Fang Zhangsheng, Xie Jun, Wang Guangjun, Yu Deguang, Liu Banghui, Gao Xiaoyang. Research progress on artificial breeding of Japanese eel [J]. Anhui Agricultural Sciences, 2013, 41(34):13269-13272), and eel breeding is still very difficult.

[0003] Currently, China mainly references Japan's artificial spawning breeding technology. While this technology can achieve eel spawning and artificial insemination, the success rate of induced spawning is low, and all hatched larvae die before completing their first metamorphosis (usually within 6-8 days). This technology suffers from problems such as asynchronous gonadal development in males and females, difficulty in controlling the timing of induced spawning, poor sperm and egg quality, low hatching rate, and short survival period for newly hatched larvae. Fish typically achieve higher gamete quality and thus higher larval survival rates through natural reproduction, but eels cannot naturally mature and mate under artificial breeding conditions.

[0004] To address the aforementioned issues, the applicant assisted in the synchronous maturation of eel broodstock gonads by adjusting the composition and dosage of the maturation-inducing drugs and combining them with habitat simulation and other technical means. This promoted natural mating of eels, overcame the shortcomings of the aforementioned artificial breeding techniques, and resulted in high-quality gametes and extended the survival time of larvae. Summary of the Invention

[0005] The purpose of this invention is to provide an artificially assisted natural reproduction method for eels, which solves the problems of low success rate of artificial spawning, low hatching rate of fertilized eggs, and short survival period of fry.

[0006] To achieve the above objectives, the present invention adopts the following technical measures:

[0007] A method for artificially assisted natural reproduction of eels includes the following steps:

[0008] I. Selection and Breeding of Broodstock

[0009] 1. Source of parent fish

[0010] Select eels of sexual maturity;

[0011] 2. Cultivation Environment

[0012] Water body: salinity 4-6‰, water temperature 20-25℃, pH 7.8~8.2, dissolved oxygen ≥7mg / L, ammonia nitrogen ≤0.5 mg / L, nitrite nitrogen ≤0.1 mg / L, avoid strong light, maintain a slight flow of water;

[0013] Nurturing ponds: Circular or oval cement ponds, 12-18㎡ in area, 0.5-1.5m in depth, with male and female fish stocked together, at a density not exceeding 5 fish / m². 3 The bottom is covered with fine sand, PVC pipes are installed as concealment, and shade netting is used to reduce stress.

[0014] 3. Pre-breeding nutritional fortification

[0015] Add 12-15% high-protein feed to the eel's basic feed, along with 0.1-0.3g / kg of vitamin E and 3-7% fish oil. The daily feeding amount is 5-8% of the eel's body weight, divided into two feedings in the morning and evening. The breeding period is a minimum of 1 month and a maximum of 2 months.

[0016] II. Simulation of Breeding Environment and Induced Maturation

[0017] 1. Environmental Simulation

[0018] Breeding ponds: Circular or oval cement ponds, 2-5 square meters in area, 1.0-1.5 meters deep, with male and female fish kept separately, and a stocking density not exceeding 5 fish / m². 3 Install PVC pipes as concealment and cover with shade netting to reduce stress;

[0019] Water temperature: Gradually decrease from 20-25℃ to 18℃, decreasing by 0.4-0.6℃ per day, and keep it stable;

[0020] Lighting: Use low light (50-100 lux), with a light cycle of 5-7 hours of light and 17-19 hours of darkness;

[0021] Salinity: Maintain a stable salinity of 30-35‰, change 30%-50% of the water weekly, and use sand filtration to avoid drastic fluctuations in salinity;

[0022] Water flow: Use a small water pump to flush the water and maintain a slight flow to simulate an ocean current environment, stimulating the parent fish to mate;

[0023] Water quality management: Dissolved oxygen ≥7mg / L, ammonia nitrogen ≤0.5mg / L, nitrite ≤0.1mg / L, pH 7.8~8.2;

[0024] 2. Hormone-induced ripening

[0025] Female fish should be injected weekly with 4-6 µg / kg of luteinizing hormone-releasing hormone analog A3 + 0.5-1.5 mg / kg of carp pituitary extract + 80-120 IU / kg of human chorionic gonadotropin. Male fish should be injected weekly with 0.1-0.3 mg / kg of carp pituitary extract + 40-60 IU / kg of human chorionic gonadotropin. Before injection, gently squeeze the abdomen of the male fish. If semen flows out, the dosage for the male fish should be halved.

[0026] III. Natural Mating and Spawning Induction and Fertilized Egg Collection

[0027] 1. Assessment of sexual maturity

[0028] Female fish: The abdomen is noticeably swollen, and you can feel egg-like particles when you press it lightly. The vent is red, swollen, and protruding. Male fish: When you press the abdomen lightly, milky white semen flows out, and the sperm motility is ≥70%.

[0029] 2. Natural mating

[0030] A square net cage, 1.0-1.5m long, is placed in the breeding pond as an incubation frame, and PVC pipes are placed inside the net cage.

[0031] Pair male and female fish in a ratio of 1:2 to 3, place them in an incubation box, and then minimize human interference and keep the environment quiet.

[0032] Maintain a slight water flow within the incubation box;

[0033] 3. Observation of oviposition and collection of fertilized eggs

[0034] After the eels spawn, the PVC pipes and parent fish are removed, and the hatching frames are transferred to the hatching pond for incubation.

[0035] IV. Incubation of Fertilized Eggs and Fry Rearing

[0036] 1. Incubation conditions

[0037] Hatching pond: water volume not exceeding 5m 3 Water temperature 20~25℃, salinity 30~33, dissolved oxygen ≥7mg / L, daily water exchange of 20% in the hatching pond, adjust the water inflow and water inflow method to prevent fertilized eggs from rolling in the water, do not aerate during the hatching period of fertilized eggs, and after the fry hatch, nano oxygenation tubes can be placed at the bottom of the pond to increase oxygen.

[0038] Density: Not exceeding 100,000 grains / m³ 3 To avoid excessive density leading to oxygen deficiency;

[0039] Incubation time: 36-48 hours. No feeding is required after the larvae hatch and before the yolk sac disappears.

[0040] 2. Larval rearing

[0041] Start feeding: Begin feeding the fry with starter food on the 6th day after hatching;

[0042] Water quality management: Change 10% to 20% of the water daily with fresh water, maintain a stable environment, avoid light and minimize disturbance.

[0043] The method described above, preferably, involves the following environmental simulation process in the breeding pond: water temperature: gradually decreasing from 25℃ to 18℃, decreasing by 0.5℃ per day and maintaining stability; lighting: using weak light of 50~100 lux, with the lighting cycle adjusted to 6 hours of light and 18 hours of darkness.

[0044] In the preferred embodiment of the above-described method, during the hormone-induced maturation process, female fish are injected weekly with 5µg / kg of luteinizing hormone-releasing hormone analog A3 + 1mg / kg of carp pituitary extract + 100 IU / kg of human chorionic gonadotropin (HCG), while male fish are injected weekly with 0.2mg / kg of carp pituitary extract + 50 IU / kg of HCG. Before injecting the drugs, the abdomen of the male fish is gently squeezed; if semen flows out, the subsequent injection dosage for the male fish is halved.

[0045] The scope of protection of this invention also includes the application of the above-described method in the artificial breeding of eels.

[0046] Compared with the prior art, the present invention has the following advantages:

[0047] (1) Based on the physiological characteristic that the development of female eels' gonads is significantly slower than that of male eels under artificial breeding conditions, this application selects the type and dosage of ripening hormones to achieve almost synchronous development of male and female gonads, which greatly improves the breeding success rate.

[0048] (2) The hormone dosage of this invention differs significantly from the artificial spawning induction technique reported in Japan. In Japan, a ripening injection is given weekly: female fish are injected with 20 mg / kg of salmon pituitary extract (SPE), and male fish are injected with 1000 IU / kg of human chorionic gonadotropin (HCG). After approximately 11 injections, spawning is induced, followed by an injection of 2 mg / kg of DHP and artificial insemination. In this invention, female fish are injected weekly with 5 µg / kg of luteinizing hormone-releasing hormone analogue A3 (LHRH-A3) + 1 mg / kg of carp pituitary extract (CPE) + 100 IU / kg of human chorionic gonadotropin (HCG). This significantly reduces the amount of hormones used and achieves the purpose of ripening through the synergistic effect of other hormones, resulting in higher quality gametes.

[0049] (3) To address the issue of male fish maturing earlier than female fish, this invention involves injecting male fish weekly with 0.2 mg / kg of carp pituitary extract (CPE) and 50 IU / kg of human chorionic gonadotropin (HCG). Compared to existing Japanese technology, this invention uses an extremely low dose of HCG while simultaneously supplementing with a low dose of CPE. This slowly promotes the development of the male fish's gonads while avoiding endocrine disorders, thereby achieving synchronization with the female fish's development and obtaining high-quality sperm. If, due to individual differences, some male fish mature slightly earlier than female fish, the hormone level in the male fish is maintained by halving the dosage, allowing it to remain in place without ejaculating or experiencing developmental regression.

[0050] (4) Compared with the disclosed Japanese breeding method, the present invention increases the gonadal development synchronization rate of eel parent fish from 18% to 66%, the hatching rate of eel fertilized eggs from 48% to 78%, and the survival time of eel larvae from 6.8 days to 19.8 days. Detailed Implementation

[0051] Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the field; the reagents or materials described, unless otherwise specified, are all from commercial sources. The methods described in this invention are applicable to the breeding of eel species, including Japanese eel, European eel, American eel, Australian eel, bicolor eel, clouded eel, and spotted eel. This invention uses the Japanese eel as an example to illustrate the breeding method. The PVC pipe used in this invention is 0.7m long and 16cm in diameter. All hormones used are products of Ningbo No. 2 Hormone Factory. The basic feed for eel broodstock is commercial eel feed, purchased from Fujian Tianma Feed Co., Ltd.

[0052] Example 1:

[0053] A method for artificially assisted natural reproduction of eels includes the following steps:

[0054] I. Selection and Breeding of Broodstock

[0055] 1. Source of parent fish

[0056] Choose wild-caught Japanese eels with high maturity (females weighing 0.7-1.0 kg and males 0.4-0.5 kg), or individuals that have reached sexual maturity through artificial breeding.

[0057] 2. Cultivation Environment

[0058] Water requirements: salinity 5‰, water temperature 25℃, pH 7.8~8.2, dissolved oxygen ≥7mg / L, ammonia nitrogen ≤0.5 mg / L, nitrite nitrogen ≤0.1 mg / L, avoid strong light, maintain a slight flow of water.

[0059] Nursing pond: Circular or oval cement pond, 15㎡ in area, 1m deep, male and female fish are stocked together, with a stocking density not exceeding 5 fish / m². 3 The bottom is covered with fine sand, PVC pipes are installed as concealment, and shade netting is used to reduce stress.

[0060] 3. Pre-breeding nutritional fortification

[0061] Add 10% high-protein feed to the eel's basic feed, including squid, krill, sandworm, etc., and add 0.2g / kg vitamin E and 5% fish oil. The daily feeding amount is 5-8% of the body weight, divided into two feedings in the morning and evening.

[0062] Breeding period: minimum 1 month, maximum 2 months to avoid delaying the breeding time.

[0063] II. Simulation of Breeding Environment and Induced Maturation

[0064] After pre-breeding rearing, the broodstock can be transferred to the breeding pond to begin induced maturation and natural mating, spawning, and fertilization. A combination of simulated breeding environment and exogenous hormones is used to promote synchronized gonadal development and estrus initiation in the broodstock, as detailed below:

[0065] 1. Environmental Simulation

[0066] Breeding pond: Circular or oval cement pond, 3 square meters in area, 0.8 meters in depth, male and female fish are kept separately, with a stocking density not exceeding 5 fish / m². 3 Install PVC pipes as concealment and cover them with shade netting to reduce stress.

[0067] Water temperature: Gradually decrease from 25℃ to 18℃, decreasing by 0.5℃ per day, and keep it stable.

[0068] Lighting: Use low light (50~100 lux), and adjust the light cycle to 6L:18D (6 hours of light, 18 hours of darkness).

[0069] Salinity: Maintain a stable level of 30-35‰, change the water regularly (30%-50% of the water volume per week), use sand to filter seawater, and avoid drastic fluctuations in salinity.

[0070] Water flow: Use a small water pump to flush the water and maintain a slight flow (flow velocity 0.1~0.2m / s) to simulate an ocean current environment and stimulate the parent fish to mate.

[0071] Water quality management: Dissolved oxygen ≥7mg / L, ammonia nitrogen ≤0.5mg / L, nitrite ≤0.1mg / L, pH 7.8~8.2. Monitor the above water quality indicators daily, remove protein from the water regularly using a protein skimmer, and improve the water quality through activated carbon filtration or microbial agents.

[0072] 2. Hormone-induced ripening

[0073] To promote synchronized gonadal development in male and female fish, exogenous hormones are used for induction, depending on the degree of difference in gonadal development between the sexes. Female fish are injected weekly with 5µg / kg of luteinizing hormone-releasing hormone analogue A3 (LHRH-A3) + 1mg / kg of carp pituitary extract (CPE) + 100 IU / kg of human chorionic gonadotropin (HCG). Male fish are injected weekly with 0.2mg / kg of carp pituitary extract (CPE) + 50 IU / kg of HCG. Before injection, gently squeeze the abdomen of the male fish; if semen flows out, the subsequent injection dosage for the male should be halved.

[0074] III. Natural Mating and Spawning Induction and Fertilized Egg Collection

[0075] 1. Assessment of sexual maturity

[0076] Female fish: The abdomen is obviously swollen, and you can feel the eggs when you press it lightly. The vent is red, swollen and protruding. Male fish: When you press the abdomen lightly, milky white semen will flow out. The sperm motility is ≥70% (microscopic observation).

[0077] 2. Natural mating

[0078] A 1.2m long square net cage (100 mesh, used as an incubation frame) is placed in the breeding pond, and 0.7m long and 16cm diameter PVC pipes are placed in the net cage as needed.

[0079] Pair male and female fish in a ratio of 1:2 to 3, place them in an incubation box, and then minimize human interference and keep the environment quiet.

[0080] A small water pump (15W) is placed in the incubation box, and water is kept flowing from the pump (flow rate 0.05 m / s) to induce mating behavior by slightly changing the water flow rate.

[0081] 3. Observation of oviposition and collection of fertilized eggs

[0082] Spawning usually occurs at night or in the early morning; check the spawning situation every morning. Collect the eggs promptly after spawning and transfer them to the hatching pond. Alternatively, the parent fish and PVC pipes can be removed, leaving the fertilized eggs in the net cage for incubation.

[0083] IV. Incubation of Fertilized Eggs and Fry Rearing

[0084] 1. Incubation conditions

[0085] Hatching pond: water volume not exceeding 5m 3The water temperature should be 20-25℃, salinity 30-33, and dissolved oxygen ≥7mg / L. Continuously add fresh water to the hatching tank, changing 20% ​​of the water daily, adjusting the inflow rate and method to prevent fertilized eggs from rolling around in the water. Do not aerate the tank during incubation; after the fry hatch, use nano-aeration tubes placed at the bottom of the tank for oxygenation.

[0086] Density: Not exceeding 100,000 grains / m³ 3 To avoid excessive density leading to oxygen deficiency.

[0087] Incubation time: 36-48 hours. No feeding is required after the larvae hatch and before the yolk sac disappears.

[0088] 2. Larval rearing

[0089] Opening rearing: Start feeding the larvae with starter feed on the 6th day after hatching (Tanaka H, ​​Kagawa H, Ohta H. Production of leptocephali of Japanese eel (Anguilla japonica) in captivity. Aquaculture, 2001, 201(1-2):51-60.).

[0090] Water quality management: Change 10% to 20% of the water daily with fresh water, maintain a stable environment, avoid light and minimize disturbance.

[0091] The synchronization rate is calculated as follows: Synchronization rate =

[0092] The hatching rate is calculated as follows: Hatching rate =

[0093] Using the above-described breeding method, compared with previously disclosed breeding methods, the results are as follows:

[0094]

[0095] Note: The control group used artificial induced spawning breeding technology for Japanese eels (Tanaka H, ​​Kagawa H, Ohta H. Production of leptocephali of Japanese eel (Anguilla japonica) in captivity. Aquaculture, 2001, 201(1-2):51-60.), while the experimental group used the artificially assisted natural reproduction technology employed in this invention. SD represents the standard deviation.

Claims

1. A method for artificially assisted natural reproduction of eels, comprising the following steps: I. Selection and Breeding of Broodstock Water body: salinity 4-6‰, water temperature 20-25℃, pH 7.8~8.2, dissolved oxygen ≥7mg / L, ammonia nitrogen ≤0.5 mg / L, nitrite nitrogen ≤0.1 mg / L, avoid strong light, maintain a slight flow of water; Nurturing ponds: 12-18㎡ in area, 0.5-1.5m in depth, where sexually mature male and female eels are stocked together at a density not exceeding 5 eels / m². 3 The bottom is covered with fine sand, PVC pipes are installed as concealment, and shade netting is used to cover it. Add 12-15% high-protein feed to the eel's basic feed, along with 0.1-0.3g / kg of vitamin E and 3-7% fish oil. The daily feeding amount is 5-8% of the eel's body weight, divided into two feedings in the morning and evening. The breeding period is a minimum of 1 month and a maximum of 2 months. II. Simulation of Breeding Environment and Induced Maturation Breeding pond: 2-5㎡ in area, 1.0-1.5m in depth, male and female fish are kept separately, with a stocking density not exceeding 5 fish / m². 3 Install PVC pipes as concealment and cover them with shade netting; Water temperature: Gradually decrease from 20-25℃ to 18℃, decreasing by 0.4-0.6℃ per day; Light intensity: 50~100 lux, light cycle adjusted to 5-7 hours of light and 17-19 hours of darkness; Salinity: kept stable at 30-35‰, with a weekly water change rate of 30%-50%. Water flow: Maintaining a slight flow in the water body; Water quality management: Dissolved oxygen ≥7mg / L, ammonia nitrogen ≤0.5mg / L, nitrite ≤0.1mg / L, pH 7.8~8.2; Female fish should be injected weekly with 4-6 µg / kg of luteinizing hormone-releasing hormone analog A3 + 0.5-1.5 mg / kg of carp pituitary extract + 80-120 IU / kg of human chorionic gonadotropin. Male fish should be injected weekly with 0.1-0.3 mg / kg of carp pituitary extract + 40-60 IU / kg of human chorionic gonadotropin. Before injection, gently squeeze the abdomen of the male fish. If semen flows out, the dosage for the male fish should be halved. After the male and female fish reach sexual maturity, they should be paired in a ratio of 1:2 to 3 and placed in an incubation box. Place a PVC pipe in the incubation box, maintain a slight water flow, and wait for them to lay eggs. III. Incubation of Fertilized Eggs and Fry Rearing Hatching pond: water volume not exceeding 5m 3 Water temperature 18~20℃, salinity 30~33, dissolved oxygen ≥7mg / L, daily water exchange of 20% in the hatching pond, avoid fertilized eggs rolling in the water, do not aerate during the hatching period, and add oxygen after the fry hatch. Density: Not exceeding 100,000 grains / m³ 3 ; Incubation time: 36-48 hours; Start feeding: Start feeding the fry with starter food on the 6th day after hatching.

2. The method according to claim 1, characterized in that, During the environmental simulation process, the breeding tank water temperature was gradually reduced from 25℃ to 18℃, decreasing by 0.5℃ daily and remaining stable; micro-flow water stimulation was also applied. Lighting: Use low light of 50-100 lux, with a light cycle of 6 hours of light and 18 hours of darkness.

3. The method according to claim 1, characterized in that, During the hormone-induced maturation process, female fish are injected weekly with 5µg / kg of luteinizing hormone-releasing hormone analog A3 + 1mg / kg of carp pituitary extract + 100 IU / kg of human chorionic gonadotropin (hCG), while male fish are injected weekly with 0.2mg / kg of carp pituitary extract + 50 IU / kg of hCG. Before injection, the abdomen of the male fish is gently squeezed; if semen flows out, the dosage for subsequent injections to the male fish is halved.

4. The application of the method described in claim 1 in the artificial breeding of eels.

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