An artificial assisted natural reproduction method for eel

Abstract

The application belongs to the field of aquaculture, and discloses an artificial assisted natural breeding method of eel, aiming at the problems of different development of female and male gonads, difficult control of spawning time, short survival period of initial hatched fry and the like in the artificial breeding technology of eel, the application adjusts the type and dosage of maturation drugs, and combines habitat simulation and other technical means to assist the synchronous maturation of gonads of eel parent fish, promote the natural mating of eel, and solve the defects of the above artificial breeding technology. Compared with the disclosed breeding technology, the application, through the synergistic effect of hormones and the habitat simulation technology, significantly reduces the dosage of pituitary and chorionic gonadotropin while ensuring the maturation effect, reduces the physiological stress of parent fish, and obtains high-quality gametes, so that the synchronous rate of gonad development of eel parent fish is increased from 18% to 66%, the hatching rate of eel fertilized eggs is increased from 48% to 78%, and the survival time of eel fry is prolonged from 6.8 days to 19.8 days.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of aquaculture, and particularly relates to a method for artificially assisting natural reproduction of eel. BACKGROUND

[0002] Artificial breeding of eel (including Japanese eel, European eel, American eel and all species of Anguilla) has been a world problem. Artificially bred eels cannot mature and spawn naturally, and therefore artificial induction of gonadal maturation and spawning and fertilization technology must be developed. At present, only Japan has reported successful full artificial reproduction of 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). At present, scholars in various countries can not achieve ideal results by referring to this technology (Qiu J X, Fang Z S, Xie J, Wang G J, Yu D G, Liu B H, Gao X Y. Research progress of artificial reproduction of Japanese eel (Anguilla japonica) [J]. Anhui Agricultural Sciences, 2013, 41 (34): 13269-13272), and eel reproduction is still difficult.

[0003] At present, the main artificial spawning reproduction technology in China refers to that of Japan, which can achieve eel spawning and artificial insemination, but has low success rate of induced spawning, and all the membrane larvae die before completing the first metamorphosis development (usually within 6-8 days). This technology has problems such as asynchronous development of female and male gonads, difficulty in grasping the time of induced spawning, poor quality of sperm and egg, low hatching rate, and short survival period of initial hatched larvae. Natural reproduction of fish can obtain high quality gametes and thus high survival rate of larvae, but the gonads of eel cannot mature naturally and mate under artificial breeding conditions.

[0004] In view of the above problems, the applicant adjusts the composition and dosage of the gonad maturation drug of parent fish, and combines habitat simulation and other technical means to assist synchronous maturation of eel parent gonads, promote natural mating of eel, and solve the defects of the above artificial breeding technology, so as to obtain high quality gametes and prolong the survival time of larvae. SUMMARY

[0005] The present application provides a method for artificially assisting natural reproduction of eel, which solves the problems of low success rate of induced spawning of eel, low hatching rate of fertilized eggs, and short survival period of larvae.

[0006] In order to achieve the above purpose, the following technical measures are taken in the present application:

[0007] A method for artificially assisting natural reproduction of eels, comprising the following steps:

[0008] I. Selection and cultivation of parent fish

[0009] 1. Source of parent fish

[0010] Select individuals of the sexual maturity age of eels;

[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, keep slight flow of water;

[0013] Cultivation pool: circular or oval cement pool, area 12-18㎡, water depth 0.5-1.5m, female and male fish are raised together, the stocking density is not more than 5 tails / m 3 , the bottom is paved with fine sand, PVC pipes are set as hiding places, and sunshade nets are covered to reduce stress;

[0014] 3. Nutritional enhancement before reproduction

[0015] Add 12-15% high-protein bait, 0.1-0.3g / kg vitamin E, and 3-7% fish oil in eel basic feed, the daily feeding amount is 5-8% of body weight, feeding twice a day, and the cultivation period is at least 1 month and not more than 2 months;

[0016] II. Simulation of breeding environment and maturation

[0017] 1. Environmental simulation

[0018] Breeding pool: circular or oval cement pool, area 2-5㎡, water depth 1.0-1.5m, female and male fish are raised separately, the stocking density is not more than 5 tails / m 3 , PVC pipes are set as hiding places, and sunshade nets are covered to reduce stress;

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

[0020] Light: use weak light 50~100lux, adjust the light cycle to 5-7 hours of light and 17-19 hours of darkness;

[0021] Salinity: stabilize at 30~35‰, water exchange 30%~50% per week, use sand-filtered seawater, and avoid sharp fluctuations in salinity;

[0022] Water flow: use a small water pump to flush water, keep the water slightly flowing to simulate ocean current environment, and stimulate the spawning of parent fish;

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

[0024] 2. Hormone ripening

[0025] Female fish are injected with luteinizing hormone analogue A3 4-6 μg / kg + carp pituitary extract 0.5-1.5 mg / kg + chorionic gonadotropin 80-120 IU / kg every week, male fish are injected with carp pituitary extract 0.1-0.3 mg / kg + chorionic gonadotropin 40-60 IU / kg every week, and the male fish's abdomen is gently squeezed before injection. If semen is discharged, the dose of the male fish is halved;

[0026] III. Induction of natural spawning and collection of fertilized eggs

[0027] 1. Sex maturity determination

[0028] Female fish: obvious abdominal distension, light pressure with egg particles, red and swollen cloaca; Male fish: light pressure on the abdomen with white semen, sperm motility ≥ 70%;

[0029] 2. Natural mating

[0030] The breeding pool is placed with a square net cage 1.0-1.5 m long as a hatching frame, and a PVC pipe is placed in the net cage;

[0031] Female and male fish are placed in the hatching frame at a ratio of 1:2-3, and human disturbance is reduced afterwards to maintain a quiet environment;

[0032] Maintain a slight water flow in the hatching frame;

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

[0034] After the eel spawns, the PVC pipe and parent fish are removed, and the hatching frame is transferred to the hatching pool for hatching;

[0035] IV. Fertilized egg hatching and larval fish cultivation

[0036] 1. Hatching conditions

[0037] Hatching pool: water body not exceeding 5 m 3 , water temperature 20-25℃, salinity 30-33, dissolved oxygen ≥ 7 mg / L, hatching pool water change 20% per day, adjust water inflow and mode to avoid fertilized eggs rolling in the water body, do not aerate during fertilized egg hatching, and use nano oxygenation tube to place at the bottom of the pool for oxygenation after the larvae hatch;

[0038] Density: not more than 100,000 particles / m 3 , avoid high density leading to hypoxia;

[0039] Incubation time: 36~48 hours, the larvae do not need to be fed after the yolk sac disappears after the membrane is broken;

[0040] 2. Larvae cultivation

[0041] Opening cultivation: start feeding the opening bait on the 6th day after the larvae are out of the membrane;

[0042] Water quality management: replace 10%~20% of fresh water every day, maintain a stable environment, avoid light and reduce disturbance.

[0043] The method described above, preferably, in the environmental simulation process, the breeding pool: the water temperature gradually decreases from 25 DEG C to 18 DEG C, decreases by 0.5 DEG C per day, and remains stable; the light intensity is 50~100 lux, and the light cycle is adjusted to 6 hours of light and 18 hours of darkness;

[0044] The method described above, preferably, in the hormone maturation process, the female fish is injected with luteinizing hormone releasing hormone analogue A3 5 mu g / kg + carp pituitary extract 1 mg / kg + chorionic gonadotropin 100 IU / kg every week, and the male fish is injected with carp pituitary extract 0.2 mg / kg + chorionic gonadotropin (HCG) 50 IU / kg every week, and the male fish is gently squeezed before injection of the drug, if there is semen, the dose of the male fish is halved.

[0045] The protection scope of the present application also includes the application of the above method in artificial breeding of eel.

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

[0047] (1) According to the physiological characteristics that the development process of the female gonad is significantly slower than that of the male gonad of eel under artificial breeding conditions, the type and dose of the maturation hormone are selected, so that the female and male gonads develop almost synchronously, and the breeding success rate is greatly improved.

[0048] (2) The hormone dose of the present application is significantly different from the artificial induction technology reported in Japan, Japan injects one maturation needle per week: female fish is injected with salmon pituitary extract (SPE) 20 mg / kg, and male fish is injected with chorionic gonadotropin (HCG) 1000 IU / kg, about 11 injections are performed before induction, and DHP 2 mg / kg is injected before artificial insemination during induction. In the present application, the female fish is injected with luteinizing hormone releasing hormone analogue A3 (LHRH-A3) 5 mu g / kg + carp pituitary extract (CPE) 1 mg / kg + chorionic gonadotropin (HCG) 100 IU / kg every week during maturation, which greatly reduces the amount of hormone used, achieves the purpose of maturation through the synergistic effect of other hormones, and can obtain higher quality gametes.

[0049] (3) For the problem of male fish maturing earlier than female fish, the male fish in the present application is injected with 0.2 mg / kg of carp pituitary extract (CPE) and 50 IU / kg of chorionic gonadotropin (HCG) every week. Compared with the prior art in Japan, the present application adopts a very low dose of HCG and simultaneously supplements a low dose of CPE, which can slowly promote the development of the gonad of the male fish while avoiding endocrine disorders in the fish body, thereby achieving the purpose of synchronizing the development of the male fish with the female fish and obtaining high-quality sperm. If due to individual differences, there are individual male fish that mature slightly earlier than female fish, the hormone level in the male fish body is maintained by halving the dose, so that the male fish waits in place without spawning or development regression.

[0050] (4) Compared with the published Japanese breeding method, the gonad development synchronization rate of eel parent fish is increased from 18% to 66%, the fertilized egg hatching rate of eel is increased from 48% to 78%, and the survival time of eel larvae is prolonged from 6.8 days to 19.8 days. DETAILED DESCRIPTION

[0051] The technical solutions described in the present application are all conventional solutions in the field unless otherwise specified; the reagents or materials described are all from commercial channels unless otherwise specified. The method described in the present application is suitable for the reproduction of Anguilla species, including Japanese eel, European eel, American eel, Australian eel, two-colored eel, clouded eel, and flower eel. The present application takes Japanese eel as an example to illustrate the breeding method. The PVC pipe used in the present application has a length of 0.7 m and a diameter of 16 cm. The hormones used are all products of Ningbo Second Hormone Factory. The basic feed for eel parent fish is a commercial eel feed purchased from Fujian Tianma Feed Co., Ltd.

[0052] Example 1

[0053] A method for artificially assisting the natural reproduction of eel, comprising the following steps:

[0054] I. Selection and cultivation of parent fish

[0055] 1. Source of parent fish

[0056] Japanese eels (female fish weighing 0.7-1.0 kg, male fish weighing 0.4-0.5 kg) with high maturity degree in the wild are selected, or individuals that have reached the age of sexual maturity through artificial breeding are selected.

[0057] 2. Cultivation environment

[0058] Water: salinity 5‰, water temperature 25℃, pH 7.8~8.2, dissolved oxygen ≥7 mg / L, ammonia nitrogen ≤0.5 mg / L, nitrite nitrogen ≤0.1 mg / L, avoid strong light, and keep the water flowing slightly.

[0059] Breeding pond: circular or oval cement pond, area 15 m2, water depth 1 m, male and female fish are raised together, the stocking density is not more than 5 tails / m 3 . Lay fine sand at the bottom, set PVC pipe as shelter, cover sunshade net to reduce stress.

[0060] 3. Nutritional enhancement before breeding

[0061] Add 10% high-protein bait to eel basic feed, the high-protein bait includes squid, phosphorus shrimp, sandworm, etc., add 0.2 g / kg vitamin E, 5% fish oil, the daily feeding amount is 5-8% of the body weight, feed twice a day.

[0062] Breeding period: minimum 1 month, not to delay the breeding time, maximum not more than 2 months.

[0063] II. Breeding environment simulation and maturation

[0064] The parent fish after pre-breeding cultivation can be transferred to the breeding pond to start maturation and natural mating, and the method of combining breeding environment simulation with exogenous hormone maturation is adopted to promote the synchronous development of the gonads of male and female parent fish, the estrus of parent fish and mating, as follows:

[0065] 1. Environment simulation

[0066] Breeding pond: circular or oval cement pond, area 3 m2, water depth 0.8 m, male and female fish are raised separately, the stocking density is not more than 5 tails / m 3 . Set PVC pipe as shelter, cover sunshade net to reduce stress.

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

[0068] Light: use weak light (50~100 lux), adjust the light cycle to 6L:18D (light for 6 hours, dark for 18 hours).

[0069] Salinity: stable at 30~35‰, regular water change (30%~50% of water change per week), use sand filter seawater to avoid sharp fluctuation of salinity.

[0070] Water flow: use small water pump to flush water, keep the water body slightly flowing (flow rate 0.1~0.2 m / s), simulate ocean current environment to stimulate the estrus of parent fish.

[0071] Water quality management: dissolved oxygen ≥7 mg / L, ammonia nitrogen ≤0.5 mg / L, nitrite ≤0.1 mg / L, pH 7.8~8.2, monitor the above water quality indexes every day, regularly remove water protein with protein separator, improve the water body through activated carbon filtration or microbial preparation.

[0072] 2. Hormone ripening

[0073] To promote the synchronous development of the gonads of male and female fish, exogenous hormones need to be used for induction according to the degree of difference in the development of the gonads of male and female fish. The female fish are injected with 5 µg / kg of LHRH-A3, 1 mg / kg of CPE, and 100 IU / kg of HCG every week, and the male fish are injected with 0.2 mg / kg of CPE and 50 IU / kg of HCG every week. Before the injection of the drugs, the abdomen of the male fish is gently squeezed, and if semen flows out, the dose of the injection of the male fish is halved.

[0074] III. Induction of spawning by natural mating and collection of fertilized eggs

[0075] 1. Judgment of sexual maturity

[0076] The female fish: the abdomen is significantly swollen, and there is a feeling of egg granules when gently pressed, and the cloaca is red and swollen; the male fish: there is a milky white semen flowing out when the abdomen is gently pressed, and the sperm motility is ≥ 70% (observed under a microscope).

[0077] 2. Natural mating

[0078] A square mesh box (mesh 100) with a length of 1.2 m is placed in the breeding pool as a hatching frame, and a PVC pipe with a length of 0.7 m and a diameter of 16 cm is placed in the mesh box as needed.

[0079] The female and male fish are placed in the hatching frame at a ratio of 1:2-3, and then human disturbance is reduced and the environment is kept quiet.

[0080] A small water pump (15 W) is placed in the hatching frame to keep the water flowing (flow rate 0.05 m / s), and the mating behavior is induced by slightly changing the water flow rate.

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

[0082] Spawning mostly occurs at night or early morning, and the spawning situation is checked every morning. The egg granules are collected in time after spawning and transferred to the hatching pool for hatching. The parent fish and the PVC pipe can also be removed, and the fertilized eggs are left in the original position of the mesh box for hatching.

[0083] IV. Hatching of fertilized eggs and cultivation of larvae

[0084] 1. Hatching conditions

[0085] Hatching pool: water body not more than 5 m 3, water temperature 20~25℃, salinity 30~33, dissolved oxygen ≥7mg / L. Fresh water is continuously added to the incubation pool, and the water exchange amount is 20% per day. The water inflow and water inflow mode are adjusted to avoid the fertilized eggs from rolling in the water body. The fertilized eggs are not aerated during incubation, and the nano oxygenation tube can be placed at the bottom of the pool to increase oxygen after the larvae hatch.

[0086] Density: no more than 100,000 particles / m 3 , to avoid high density leading to hypoxia.

[0087] Incubation time: 36~48 hours, no feeding is required after the larvae break the membrane and before the yolk sac disappears.

[0088] 2. Larvae cultivation

[0089] Opening cultivation: the larvae start to be fed with opening bait 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: 10%~20% fresh water is exchanged every day to maintain a stable environment, avoid light, and reduce disturbance.

[0091] The calculation method of the synchronization rate is: synchronization rate =

[0092] The calculation method of the incubation rate is: incubation rate =

[0093] Using the above breeding method, compared with the published breeding method, the results are as follows:

[0094]

[0095] Note: The control group is the artificial induced spawning breeding technology of Japanese eel (Japan) (Tanaka H, Kagawa H, Ohta H. Production of leptocephali of Japanese eel (Anguilla japonica) in captivity. Aquaculture, 2001, 201(1-2): 51-60.), and the experimental group is the artificial assisted natural breeding technology adopted by the present application. SD represents standard deviation.

Claims

1. An artificial assisted natural reproduction method of eel, comprising the following steps: I. Selection and cultivation of parent fish Water body: salinity 4-6‰, water temperature 20-25℃, pH 7.8~8.2, dissolved oxygen ≥7 mg / L, ammonia nitrogen ≤0.5 mg / L, nitrite nitrogen ≤0.1 mg / L, avoid strong light, keep slight water flow; Cultivation pond: area 12-18 square meters, water depth 0.5-1.5 meters, mature male and female eels are raised together, and the stocking density is not more than 5 tails / m 3 Fine sand is laid at the bottom, PVC pipes are set as shelters, and shading nets are covered. Add 12-15% high-protein bait, 0.1-0.3 g / kg vitamin E and 3-7% fish oil to the eel basic feed as eel feed, the daily feeding amount is 5-8% of the body weight, feeding twice a day, the cultivation period is at least 1 month and at most 2 months; II. Simulation of breeding environment and maturation Breeding pond: area 2-5 square meters, water depth 1.0-1.5 m, male and female fish are kept separately, and the stocking density is not more than 5 tails / m 3 , set PVC pipe as shelter, cover sunshade net; Water temperature: gradually decrease from 20-25℃ to 18℃, decrease by 0.4-0.6℃ per day; Light: 50~100lux, light cycle adjustment to 5-7 hours of light, 17-19 hours of darkness; Salinity: stable at 30~35‰, water exchange 30%~50% per week; Water flow: keep the water body slightly flowing; Water quality management: dissolved oxygen ≥7 mg / L, ammonia nitrogen ≤0.5 mg / L, nitrite ≤0.1 mg / L, pH 7.8~8.2; Inject female fish with luteinizing hormone releasing hormone analogue A3 4-6µg / kg + carp pituitary extract 0.5-1.5mg / kg + chorionic gonadotropin 80-120 IU / kg every week, inject male fish with carp pituitary extract 0.1-0.3mg / kg + chorionic gonadotropin 40-60IU / kg every week, gently press the abdomen of male fish before injection, if semen is discharged, then reduce the dose of male fish by half; Match female and male fish at a ratio of 1:2~3 after sexual maturity, put them into the incubation frame, place PVC pipes in the incubation frame, keep slight water flow, and wait for spawning; III. Hatching of fertilized eggs and cultivation of larvae Incubation pool: water volume not more than 5 m 3 , water temperature 18~20℃, salinity 30~33‰, dissolved oxygen≥7mg / L, water exchange rate of incubation pool 20% per day, avoid fertilized eggs rolling in water, no aeration during fertilized egg incubation, increase oxygen after larvae hatching; Density: no more than 100,000 particles / m 3 ; Hatching time: 36~48 hours; Opening cultivation: start feeding opening bait on the 6th day after the larvae hatched.

2. The method of claim 1, wherein, In the simulation process of the breeding environment, the breeding pool: water temperature: gradually decrease from 25℃ to 18℃, decrease by 0.5℃ per day, and keep stable; slight water flow stimulation; Light: weak light 50~100lux, light cycle adjustment to 6 hours of light, 18 hours of darkness.

3. The method of claim 1, wherein, In the maturation process, inject female fish with luteinizing hormone releasing hormone analogue A3 5µg / kg + carp pituitary extract 1mg / kg + chorionic gonadotropin 100IU / kg every week, inject male fish with carp pituitary extract 0.2mg / kg + chorionic gonadotropin 50 IU / kg every week, gently press the abdomen of male fish before injection, if semen is discharged, then reduce the dose of male fish by half.

4. The method of claim 1 in the artificial reproduction of eel.

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