Method for artificial insemination and fertilized egg incubation of wild schizothorax prenanti

Abstract

The invention provides a method for artificial insemination and fertilized egg incubation of wild schizothorax parvus, which comprises the following steps: on the basis of ascertaining the biological characteristics of the wild schizothorax parvus through tracking and monitoring for many years, capturing sexually mature male and female parent fishes, carrying out artificial insemination after sperm and egg treatment, obtaining fertilized eggs, and carrying out micro-running water incubation, so that the fertilization rate reaches 85%, and the incubation rate reaches 70%. The artificial insemination of the rare and endangered indigenous fish Schizothorax parvum in the Luagu Lake and the trial culture of the first filial generation of fish fries are successfully carried out for the first time in China, the blank in the field is filled, the artificial domestication, breeding and culture of the Schizothorax parvum are completely feasible and are preliminarily successful, and the specific fish population of the Luagu Lake is recovered, developed and cultured, so that the economic benefits of the Luagu Lake and the Schizothorax parvum are improved. High-quality offspring seeds are provided, and the method has important significance on lake enhancement and release and improvement of fishery economic benefits.

Description

Technical Field

[0001] The invention relates to the technical field of fish breeding, and in particular to a method for artificial insemination of wild schizothorax schizothorax and hatching of fertilized eggs. Background Art

[0002] Schizothorax microstomus belongs to the genus Schizothorax, subfamily Schizothorax of the family Cyprinidae. It is a rare native fish species unique to Lugu Lake in Lijiang. The dorsal fin rays iii, 7-8; anal fin rays ii, 5; pectoral fin rays i, 18-21; pelvic fin rays i, 8-10. The origin of the dorsal fin is located slightly above and in front of the origin of the pelvic fin, and the terminal unbranched fin rays are hard spines with serrations on the rear edge. The origin of the anal fin is located below and behind the end of the dorsal fin rays, and the fin rays do not reach the base of the caudal fin. The end of the pectoral fin is pointed, and the end of the fin rays reaches about the midpoint of the distance between the origins of the pectoral and pelvic fins. The origin of the pelvic fin is slightly below the origin of the dorsal fin, about the midpoint of the body length, and the end of the fin rays does not reach the origin of the anal fin. The caudal fin is forked and pointed at the end. The body is elongated, slightly flattened laterally, with a rounded abdomen, and the dorsal and ventral margins are raised to the origin of the anal scales and then become straight. The snout is pointed, the eyes are large, and the head is conical. Mouth is terminal, horseshoe-shaped, with the front end of the mouth cleft flush with the middle and lower part of the eye, the end of the premaxilla is below the nostril, the outer edge of the lower jaw is not keratinized, and the inner edge is thinner, without forming a sharp keratin edge. The lower lip only forms a narrow lateral lobe, with a smooth surface and no mastoids, the two lobes are not connected, and the posterior lip groove is discontinuous. There are two pairs of whiskers, which are small and about the same length. The snout whiskers extend to the bottom of the front edge of the nostril, and the corner whiskers extend to the bottom of the eye. There are 18-23 gill rakers on the outer side of the first gill arch; there are 3 rows of hypopharyngeal teeth: 2.3.5-5.3.2; there are 100-119 lateral line scales. The body, chest and ventral surface are all covered with fine scales, with a large axillary scale on each side of the pelvic fin base, and a row of large anal scales on each side from the anus to the anal fin base. The lateral line is complete, slightly curved in the middle and then straight into the middle of the caudal peduncle. The gill rakers are short and sparsely arranged. The hypopharyngeal bone is narrow and arc-shaped. The base of the hypopharyngeal teeth is columnar, the top is pointed, the tooth surface is spoon-shaped, and the first tooth of the main row is small. The swim bladder has two chambers, and the rear chamber is longer than the front chamber. The peritoneum is black. When alive, the dorsal and side of the body are gray-brown with many irregular black spots on them, and the abdomen is gray-white. It is a pelagic fish, omnivorous, and prefers to eat plankton. The breeding season is from June to August every year, and it goes upstream to the streams entering the lake to lay eggs.

[0003] In the past, the small-mouthed schizothorax was not only the native fish of Lugu Lake, but also the main economic fish of Lugu Lake, with an annual output of hundreds of tons. Due to the development of tourism in Lugu Lake in the last century and the introduction of alien species, small and harmful non-economic fish (small shrimp, shrimp goby, wheat ear fish, yellow croaker, Chinese minnow, etc.) were brought in. These small non-economic fish have a short growth cycle, reproduce quickly, and feed exclusively on the eggs of native fish. Especially after the mid-1980s, the introduction of Lugu Lake carp, grass carp, and crucian carp quickly formed a dominant population in Lugu Lake, which seriously affected the lake ecological environment of Lugu Lake. In addition, a hydropower station was built at the outlet of Lugu Lake, which blocked the migration channel of Lugu Lake's native fish and caused overfishing. As a result, the native fish population of Lugu Lake has dropped sharply over time, and by the beginning of this century, it has become an endangered rare species. If it is not saved in time, it will go extinct. The small-mouthed schizothorax has delicious meat and high nutritional value. It is one of the main economic fish in Lugu Lake and has high ecological, social, economic and academic value.

[0004] At present, the smallmouth schizothorax has become an uncommon species, and there is no successful precedent for artificial breeding. Breaking through the artificial breeding technology of the smallmouth schizothorax and then carrying out artificial breeding is the only effective way to protect this species. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for artificial insemination and hatching of fertilized eggs of wild small-mouthed schizothorax. On the basis of exploring the biological characteristics of wild small-mouthed schizothorax, sexually mature male and female broodstock are captured, artificial insemination is performed after sperm and egg treatment, and the fertilized eggs are transferred to a self-made incubator for micro-flow water hatching; artificial insemination of small-mouthed schizothorax and trial breeding of the first-generation fry are successfully carried out for the first time, filling the gap in this field.

[0006] The technical solution of the present invention is:

[0007] A method for artificial insemination of wild schizothorax schizothorax and hatching of fertilized eggs comprises the following steps:

[0008] Step 1: Build broodstock domestication facilities: The domestication facilities use aquaculture barrels produced by Fujian Huayu Ecological Technology Co., Ltd. The material is HDPP polypropylene food grade, and the maximum volume of the barrel is <7.06m 3 Two; Specifications: diameter 300*H100cm; Maximum volume of medium barrel <3.14m 3 Specifications: two diameters 200*H100cm; small temporary storage barrels <maximum volume 0.8m 3 , Specifications: Five diameters 80*H70cm. Each breeding tank is independently equipped with a three-layer turnover box with a length of 60cm, a width of 40cm, and a height of 66cm as a water circulation trickle filter box water circulation device.

[0009] Step 2: Collection and fishing of broodstock: In order to ensure the pure quality of the original species and the uniqueness of the broodstock traits, the fishing operation area is in the waters of Lugu Lake under the jurisdiction of Luoshui Village Committee, Yongning Town, Ninglang County. The section name and longitude and latitude are: Hongyanzi: E100°, 7509"; N27°, 7115"; Xiaoyuba: E100°, 7549"; N27°, 6983"; Dayuba: E100°, 7607"; N27°, 6920". Experienced local fishermen are invited to serve as the main catchers to carry out fishing operations. The fishing method uses a polyethylene three-layer gill net (length 50m*width 20m), mesh size: (6-8cm), ground cage (length 3m*width 0.25m*height 0.25m)

[0010] Step 3: Water injection: Connect the water pipes to the natural spring water from Lugu Lake in the breeding barrels to ensure sufficient water and fresh water quality, so as to provide a good environment for the breeding habitat. The domestication barrels are arranged in a cool and quiet room, and each breeding barrel has independent water inlet and outlet. The transparency is greater than 120cm, the dissolved oxygen is not less than 5mg / L, the pH value is 6.5-7.5, and the water temperature is 12.1-19.5℃.

[0011] Step 4: Trauma prevention and disinfection of broodstock: Small-mouthed schizothorax caught in the wild are placed in a temporary holding box on board: the volume is 80 liters, and a simple disinfection treatment is carried out with 4‰ salt and 10pp ginger solution for 1h-1.5h to reduce the aggravation of the fishing net hanging marks. The captured wild broodstock are transported to the domestication point in a temporary holding box (transportation distance 1 km), and the water temperature is adjusted to the temporary holding bucket (small bucket). After the water temperature is consistent, the fish body is smeared with gentian violet solution to disinfect the fish body hanging marks, and then placed in a temporary holding bucket for temporary holding. The next day, 450 liters of water are bathed in 4 tablets of methylene blue and 10ppm of oxytetracycline hydrochloride for prevention and treatment for 48 hours. The bath is bathed for 48 hours every two days for 3 consecutive times to try to heal the fish body hanging marks.

[0012] Step 5: Breeding and cultivation of broodstock: Choose a brooding facility with mountain spring water around Lugu Lake and the temperature is between 12.1-19.5℃ for temporary brooding and domestication. Put the caught wild smallmouth schizothorax into the broodstock brooding facility, 3-4kg per cubic meter of water. The broodstock have no desire to eat when they are just caught. After a few days, the broodstock have no traces of being caught by the gillnet and their physical condition has recovered. Try feeding them with thawed red nematodes and the results show that the broodstock show active feeding performance. In the later stage, add artificial sturgeon slow-sinking expanded feed (Sturgeon Special), particle size: 2.0, crude protein: greater than or equal to 41.0, feeding amount is 4-5% of the fish body weight, and feed once in the morning and evening every day.

[0013] Step 6: Artificial induction of spawning: After the captured small-mouthed schizothorax is transported to the taming site, the water temperature in the transport box is adjusted to the water temperature in the temporary holding barrel to keep it consistent. The water temperature in the taming barrel is maintained at 11-16°C. Then the broodstock are measured, weighed, and the sex is identified. The wounds of the broodstock are smeared with gentian violet solution and placed in a 500L temporary holding barrel for temporary fertilization. A 20mm PVC pipe is used to introduce unpolluted mountain spring water around Lugu Lake to continuously add new water to the temporary holding barrel, and an aeration pump is used to increase oxygen so that the water in the barrel is fully stirred to stimulate the development of the broodstock's gonads. The broodstock temporary holding barrel is enlarged before inducing spawning. The water inlet and outlet are exchanged every 20 to 25 minutes in the temporary holding tank. A heating rod is placed in the temporary holding tank to slowly increase the water temperature by 1°C every 24 hours until the water temperature is maintained at a constant temperature of 19.5°C. The pH is weakly alkaline, the dissolved oxygen is 6.0 to 6.4 mg / L, the salinity is 16 to 17, and the ammonia nitrogen is <0.1 mg / L. The water flow stimulation is continuously increased so that both male and female fish reach a similar stage of gonad maturity. The sexual maturity of the broodstock is observed at any time. If male and female fish are found chasing each other, artificial insemination is carried out when the male-to-female ratio reaches 1:2.

[0014] Step 7: Artificial insemination: Dry insemination is used. First, the equipment is disinfected. The ratio of female fish to male fish is 1:2-7. The eggs and sperm of sexually mature male and female schizothorax are squeezed into the prepared container from their abdomens. Gently stir with chicken feathers for 30 seconds to fully mix the two. After standing for 5 minutes, add appropriate amount of clean water to wash the fertilized eggs, preferably to submerge the sperm eggs. Continue stirring for 20 seconds, and then wash with the prepared clean water 3 times to obtain fertilized eggs. The diameter of the fertilized eggs before absorbing water is 2.3-2.9mm, and the diameter of the eggs after absorbing water is 2.8-3.3mm. The fertilized eggs obtained are incubated on site (Lugu Lake domestication site).

[0015] Step 8: Artificial incubation: The fertilized eggs are placed in a pre-numbered homemade open cage in a bucket for incubation. The homemade open cage device is made of 30-40 mesh mesh (specifications: 54x400x17cm). The cage device used is soaked in 80ml of potassium permanganate solution per liter of water for 24 hours a few days in advance and then exposed to the sun for disinfection and sterilization before use. Each incubator is placed with 3000-4000 fertilized eggs, and the water depth of the incubator is controlled at 15-20cm. During the incubation period, dead eggs and residues are sucked out with a plastic straw at any time to keep the water quality in the incubator fresh and sufficient dissolved oxygen to prevent the occurrence of diseases and pests such as saprolegniasis; the fertilized eggs are incubated with natural spring water (water temperature: 14℃-16℃) and artificial temperature control (18℃-19℃) for comparative experimental incubation. Artificial temperature control is achieved by placing a heating rod (brand: SenSen intelligent fish tank heating rod; model: URB-1000; temperature control range: 20-35℃; heating tube material: explosion-proof quartz tube) to make the hatching water temperature rise slowly by 1℃ every 24 hours until the water temperature is maintained at a constant temperature of 18℃-19℃ for incubation under artificial temperature control conditions such as circulating water aeration and sterilization. The fertilized eggs reach the middle gastrula after 54 hours, and begin to hatch after 119 hours. The hatching is completed after 153 hours, and the fry are 8.79-9.53mm long; after 217 hours, the fry are 9.62-10.45mm long and start to swim and feed. They are fed with Artemia nauplii 4 times a day. Every 10,000 fry are fed with 20g of nauplii hatched from Artemia eggs every day. Adjust the feeding amount according to the eating conditions of the fry; when the fry are about 3 cm long, put the fry in the homemade hatchery into a circulating breeding barrel with a diameter of 2-3m and feed them with high-protein artificial feed (high-end marine fish fry microparticle compound feed, particle size: S1, crude protein: greater than or equal to 52). Feed 4 times a day, and the feeding amount is 3% of the fry's body weight. Check the growth status of the fry under a microscope every morning. If parasites and other pests are found, use drugs to prevent and treat them in time. The following conclusions were drawn from comparative hatching experiments: natural spring water (water temperature: 14℃-16℃) was used for incubation of fertilized eggs, and the time for fertilized eggs to hatch was 216h-256h; artificial temperature control was used for incubation, and the time for fertilized eggs to hatch was 119h-153h; the growth rates of fry hatched from the same batch of fertilized eggs on the same day were compared after 29 days of cultivation: the fry hatched under spring water temperature of 14℃-16℃ grew to 13mm in length, and the fry hatched under artificial temperature control water temperature of 18℃-19℃ grew to 23mm in length. The growth rate of fry hatched under artificial temperature control conditions was more than twice that of fry cultivated under natural water temperature conditions.

[0016] Preferably, the fishing net is a polyethylene three-layer barbed net, the net is woven with double knots, and the mesh is used horizontally. The upper line of the net is equipped with foam plastic floats at certain intervals, and the lower line is equipped with lead sinkers.

[0017] Preferably, the collection and fishing of the broodstock is to ensure the pure line quality of the original species and the uniqueness of the broodstock traits. The fishing operation area is in the waters of Lugu Lake under the jurisdiction of Luoshui Village Committee, Yongning Town, Ninglang County. The section name and longitude and latitude are: Hongyanzi: E100°, 7509"; N27°, 7115"; Xiaoyuba: E100°, 7549"; N27°, 6983"; Dayuba: E100°, 7607"; N27°, 6920". Local fishermen with rich experience are invited to serve as the main catcher to carry out fishing operations. The fishing method uses a polyethylene three-layer gill net (length 50m*width 20m), mesh size: (6-8cm), and a ground cage (length 3m*width 0.25m*height 0.25m). Grasping the broodstock fishing time, fishing nets, mesh size, and fishing location selection are the key to collecting broodstock.

[0018] Preferably, during the artificial induction of spawning of the broodstock, new water is continuously added to the temporary holding barrel with natural spring water from Lugu Lake, and oxygen is added with an aerator pump, so that the water in the barrel is fully stirred to stimulate the gonadal development of the broodstock, and the water in and out of the temporary holding barrel for the broodstock is increased before inducing spawning, and the temporary holding barrel is exchanged every 20 to 25 minutes. A heating rod is placed in the temporary holding barrel to gradually increase the water temperature by 1°C every 24 hours until the water temperature is maintained at a constant temperature of 19.5°C. By increasing the water flow rate and controlling the temperature, both male and female fish reach a similar gonadal maturity period, creating conditions for obtaining fertilized eggs through artificial insemination.

[0019] Preferably, a comparative experiment is conducted between the artificial temperature control incubation method and the natural water temperature incubation method: the artificial temperature control (water temperature: 18°C-19°C) heating rod heating incubation method is better than the natural spring water (water temperature: 14°C-16°C) incubation method, which is mainly reflected in the differences in the health status and growth rate of the fry.

[0020] Preferably, the hatched fry are fed with Artemia nauplii when they start to swim horizontally and feed, Artemia nauplii are fed 4 times a day, and 20g Artemia nauplii hatched from Artemia eggs are fed to every 10,000 fry every day. The feeding amount is adjusted according to the fry's eating situation; when the fry is about 3cm long, the fry in the homemade hatching box is placed in a 2-3m diameter circulating breeding barrel for feeding, and high-protein artificial feed (high-grade marine fry microparticle compound feed, particle size: S1, crude protein: greater than or equal to 52) is fed 4 times a day, and the feeding amount is 3% of the fry's body weight, which solves the problem of fry starting to eat and switching to later food.

[0021] The beneficial effects of the present invention are:

[0022] (1) For the first time, artificial insemination, hatching, and fry cultivation of wild smallmouth schizothorax from Lugu Lake were successfully carried out, filling a gap in this field. A series of technical problems such as the fishing time, fishing nets, fishing techniques, broodstock transportation, broodstock domestication, and fish disease prevention of the broodstock of smallmouth schizothorax, a rare indigenous fish species unique to Lugu Lake, were explored, so that the broodstock can survive to a greater extent after being captured, thus overcoming the problems of the difficulty in catching smallmouth schizothorax and the high mortality rate of broodstock due to serious injuries during the fishing process.

[0023] (2) We have found effective and feasible technical methods such as artificial induction of labor and artificial insemination, making artificial insemination more reasonable and scientific, thus overcoming the defect of low fertilization rate of small-mouthed schizothorax.

[0024] (3) The incubation of fertilized eggs uses a circulating water system to avoid interference from external factors, which is conducive to the large-scale breeding and cultivation of small-mouthed schizothorax and the promotion of large-scale pond farming.

[0025] (4) The feeding method of starter feed and artificial feed to the fertilized eggs of small-mouthed schizothorax after hatching has improved the survival rate of the fry. For the first time, the first generation of fry of small-mouthed schizothorax, a rare indigenous fish of Lugu Lake, was bred. The bred fry were used to increase the amount of indigenous fish resources in Lugu Lake through artificial propagation and release, which brought hope for the protection of endangered rare species in Lugu Lake that are about to face extinction. At the same time, it provided an important reference for the artificial breeding of indigenous fish in Jinsha River.

[0026] (5) The present invention has the advantages of low cost, simple operation, high fertilization rate, hatching rate and fry survival rate. From now on, the indigenous rare and endangered schizothorax fish of Lugu Lake can be saved, and the restoration and rejuvenation of the population has become a reality. This not only effectively protects the biodiversity of Lugu Lake, but also has practical and far-reaching significance for improving people's living standards and promoting local economic development. DETAILED DESCRIPTION

[0027] 1. A method for artificial insemination of wild schizothorax schizothorax and hatching of fertilized eggs, characterized in that it comprises the following steps:

[0028] Step 1: Build broodstock rearing facilities: The rearing facilities use aquaculture barrels made of food-grade HDPP polypropylene, two large barrels with a maximum volume of <7.06m 3 , Specifications: diameter 300*H100cm; two medium-sized barrels, maximum volume <3.14m 3 Specifications: diameter 200*H100cm; five small-sized temporary storage barrels, with a maximum volume of <0.8m 3 , Specifications: diameter 80*H70cm; Each breeding tank is independently equipped with a three-layer turnover box with a length of 60cm, a width of 40cm and a height of 66cm as a water circulation trickle filter box water circulation device;

[0029] Step 2: Collection and fishing of broodstock: In order to ensure the pure quality of the original species and the uniqueness of the broodstock traits, the fishing operation area is in the waters of Lugu Lake under the jurisdiction of Luoshui Village Committee, Yongning Town, Ninglang County. The section name and longitude and latitude are: Hongyanzi: E100°, 7509"; N27°, 7115"; Xiaoyuba: E100°, 7549"; N27°, 6983"; Dayuba: E100°, 7607"; N27°, 6920". Experienced local fishermen are invited to serve as the main catchers to carry out fishing operations. The fishing method uses a polyethylene three-layer gill net (length 50m*width 20m), mesh: (6-8cm), and a ground cage (length 3m*width 0.25m*height 0.25m) for fishing;

[0030] Step 3: Water injection: Connect the water pipes to the breeding barrels to introduce mountain spring water, keep the water volume sufficient and fresh, and provide good environmental conditions for the breeding habitat. The domestication barrels are arranged in a cool and quiet room, with independent water inlet and outlet for each breeding barrel. The transparency is greater than 120cm, the dissolved oxygen is not less than 5mg / L, the pH value is 6.5-7.5, and the water temperature is 12.1-19.5℃;

[0031] Step 4: Trauma prevention and disinfection of broodstock: smallmouthed schizothorax caught in the wild are placed in a temporary holding box on board: the volume is 80L, and a simple disinfection treatment is carried out with 4‰ salt and 10pp ginger solution for 1h-1.5h to reduce the aggravation of the fishing net hanging marks. The captured wild broodstock are transported to the domestication point in a temporary holding box (transportation distance 1km), and the water temperature is adjusted to the same as the water temperature of the temporary holding small bucket. The fish body is disinfected by applying gentian violet solution to the gillnet hanging marks, and then placed in a temporary holding bucket for temporary holding; the next day, 450L of water is bathed in 4 tablets of methylene blue and 10ppm of oxytetracycline hydrochloride for prevention and treatment for 48h, and the bath is bathed for 48h every two days for 3 times in a row to try to heal the fish body hanging marks;

[0032] Step 5: Breeding and cultivation of broodstock: When broodstock are first caught, they have no desire to eat. After several days, the marks of gillnet on the broodstock’s body and their physical condition recover. Try feeding them with thawed red nematodes. The results show that the broodstock actively eat. Then add artificial sturgeon slow-sinking expanded feed (Sturgeon Special) with particle size of 2.0 and crude protein of 41.0 or more.

[0033] Step 6: Artificial induction of spawning: After the captured small-mouthed schizothorax is transported to the taming site, the water temperature in the transport box is adjusted to the water temperature in the temporary holding barrel to keep it consistent. The water temperature in the taming barrel is maintained at 11-16°C. Then the broodstock are measured, weighed, and the sex is identified. The wounds of the broodstock are smeared with gentian violet solution and placed in a 500L temporary holding barrel for temporary fertilization. A 20mm PVC pipe is used to introduce unpolluted mountain spring water around Lugu Lake to continuously add new water to the temporary holding barrel, and an aeration pump is used to increase oxygen so that the water in the barrel is fully stirred to stimulate the development of the broodstock's gonads. The broodstock temporary holding barrel is enlarged before inducing spawning. The water in and out volume is changed every 20-25 minutes in the temporary tank. A heating rod is placed in the temporary tank to gradually increase the water temperature by 1°C every 24 hours until the water temperature is maintained at a constant temperature of 19.5°C. The pH is slightly alkaline, the dissolved oxygen is 6.0-6.4 mg / L, the salinity is 16-17, and the ammonia nitrogen is <0.1 mg / L. The water flow stimulation is continuously increased to make the male and female fish reach a similar gonadal maturity period. The sexual maturity of the parent fish is observed at any time. When the male and female fish are found chasing each other, artificial insemination is carried out when the ratio of male to female reaches 1:2.

[0034] Step 7: Artificial insemination: dry insemination is adopted. First, the instruments are disinfected. The ratio of female fish to male fish is 1:2-7. The eggs and sperms of sexually mature male and female schizothorax are squeezed into the prepared vessels from their abdomens, and the two are stirred gently with chicken feathers for 30 seconds to fully mix them. After standing for 5 minutes, add appropriate amount of clean water to wash the fertilized eggs, preferably to submerge the sperm eggs, continue stirring for 20 seconds, and then wash them with the prepared clean water for 3 times to obtain fertilized eggs. The diameter of the fertilized eggs before absorbing water is 2.3-2.9mm, and the diameter of the eggs after absorbing water is 2.8-3.3mm. The fertilized eggs obtained are hatched on site (Lugu Lake domestication point);

[0035] Step 8: Artificial incubation: The fertilized eggs are placed in a pre-numbered homemade open cage in a bucket for incubation. The homemade open cage device is made of 30-40 mesh mesh with specifications of 54x400x17cm. The cage device used is soaked in 80ml of potassium permanganate solution per liter of water for 24 hours a few days in advance and then exposed to the sun for disinfection and sterilization before use. Each incubator is placed with 3000-4000 fertilized eggs. The water depth of the incubator is controlled at 15-20cm. During the incubation period, dead eggs and residues are sucked out with a plastic straw at any time to make the water quality in the incubator Keep it fresh and with sufficient dissolved oxygen to prevent the occurrence of diseases and pests such as Saprolegniasis; natural spring water is used for incubation of fertilized eggs, with a water temperature of 14℃-16℃, and the temperature of incubation and artificial temperature control is 18℃-19℃ for comparative experimental incubation; artificial temperature control is achieved by placing a heating rod (brand: Sensen intelligent fish tank heating rod; model: URB-1000; temperature control range: 20-35℃; heating tube material: explosion-proof quartz tube) to control the incubation water temperature at 18℃-19℃ for incubation under artificial temperature control conditions such as circulating water oxygenation and sterilization. The fertilized eggs reach the mid-gastrula in 54 hours and start to incubate after 119 hours. The fry started to hatch, and the hatching was completed in 153 hours. The fry was 8.79-9.53mm long. After 217 hours, the fry was 9.62-10.45mm long and began to swim and feed. They were fed with Artemia nauplii 4 times a day. Every 10,000 fry were fed with 20g Artemia nauplii hatched from eggs every day. The feeding amount was adjusted according to the fry's eating situation. When the fry was about 3cm long, the fry in the homemade hatching box was placed in a circulating breeding barrel with a diameter of 2-3m and fed with high-protein artificial feed. The feeding amount was 3 times a day, and the feeding amount was 3% of the fry's body weight. Every morning, the fry was examined under a microscope. Check the growth of fry, if parasites and other pests are found, use medicines to prevent and treat them in time; natural spring water is used for hatching fertilized eggs, the water temperature is 14℃-16℃, and the fertilized eggs take 216h-256h to hatch; artificial temperature control is used for hatching and the hatching time is 119h-153h; fry hatched from the same batch on the same day are cultivated for 29 days. The fry hatched under natural water temperature of 14℃-16℃ grow to 13mm in length, and the fry hatched under artificial temperature control of 18℃-19℃ grow to 23mm in length, which is more than twice the growth rate of fry cultivated under natural water temperature conditions.

[0036] The fishing net is a polyethylene three-layer barbed net, the net is woven with double knots and used horizontally, and the upper line of the net is equipped with foam plastic floats at certain intervals and the lower line is equipped with lead sinkers.

[0037] To ensure the pure quality of the original species and the uniqueness of the broodstock traits, the fishing operation area is in the waters of Lugu Lake under the jurisdiction of Luoshui Village Committee, Yongning Town, Ninglang County. The section name and longitude and latitude are: Hongyanzi: E100°, 7509"; N27°, 7115"; Xiaoyuba: E100°, 7549"; N27°, 6983"; Dayuba: E100°, 7607"; N27°, 6920". Local fishermen with rich experience are invited to serve as the main catchers to carry out fishing operations. The fishing method uses a polyethylene three-layer gill net (length 50m*width 20m), mesh size: (6-8cm), and a ground cage (length 3m*width 0.25m*height 0.25m).

[0038] During the artificial induction of spawning of broodstock, the captured small-mouthed schizothorax are transported to the taming site, and the water temperature in the transport box is adjusted to be consistent with the water temperature in the temporary holding barrel. The water temperature in the aquaculture barrel is maintained at 11-16°C. The broodstock are then measured, weighed, and the sex is identified. The wounds of the broodstock are smeared with gentian violet solution and placed in a 500L temporary holding barrel for temporary holding. A 20mm PVC pipe is used to introduce unpolluted mountain spring water around Lugu Lake, and new water is continuously added to the temporary holding barrel, and an aeration pump is used to increase oxygen so that the water in the barrel is fully Agitation stimulates the development of the gonads of broodstock. Before inducing spawning, increase the amount of water in and out of the temporary holding barrel for broodstock. Exchange the water every 20 to 25 minutes. Place a heating rod in the temporary holding barrel to slowly increase the water temperature by 1°C every 24 hours until the water temperature is maintained at a constant temperature of 19.5°C, with a weakly alkaline pH, 6.0 to 6.4 mg / L of dissolved oxygen, 16 to 17, and ammonia nitrogen <0.1 mg / L. Continue to increase water flow stimulation so that both male and female fish reach similar gonad maturity periods.

[0039] When the hatched fry start to swim and feed, they are fed with Artemia nauplii as training food, Artemia nauplii are fed 4 times a day, 20 g of Artemia nauplii hatched from Artemia eggs are fed to every 10,000 fry every day, and the feeding amount is adjusted according to the feeding situation of the fry; when the fry are about 3 cm long, the fry in the self-made incubator are placed in a circulation breeding barrel with a diameter of 2-3 m for feeding, and high-protein artificial feed (high-grade marine fish fry microparticle compound feed, particle size: S1, crude protein: greater than or equal to 52) is fed 4 times a day, and the feeding amount is 3% of the fry body weight.

[0040] The homemade open cage device is made of 30-40 mesh mesh with specifications of 54x400x17cm. The cage device is used after being soaked in 80ml of potassium permanganate solution per liter of water for 24 hours a few days in advance and then exposed to the sun for disinfection and sterilization before use. Each incubator box contains 3000-4000 fertilized eggs, and the water depth of the incubator box is controlled at 15-20cm.

[0041] 6. Experimental results.

[0042] A comparative experiment was conducted through artificial temperature control incubation and natural water temperature incubation: artificial temperature control, water temperature: 18℃-19℃; heating rod heating incubation method is better than natural spring water, water temperature: 14℃-16℃, incubation method, which is mainly reflected in the differences in the health status and growth rate of the fry.

[0043] Artificial hatching experiment: The fertilized eggs were placed in a pre-numbered homemade open net cage placed in a bucket for hatching. The homemade open net cage device was made of a 30-40 mesh mesh (specification: 54x400x17cm). The net cage device used was soaked in 80ml of potassium permanganate solution per liter of water for 24 hours a few days in advance and then exposed to the sun for disinfection and sterilization before use. 3000-4000 fertilized eggs were placed in each incubator, and the water depth of the incubator was controlled at 15-20cm. During the incubation period, dead eggs and residues were sucked out with a plastic straw at any time to keep the water quality in the incubator fresh and sufficient in dissolved oxygen to prevent the occurrence of diseases and insect pests such as saprolegniasis; the fertilized eggs were hatched using natural spring water (water temperature: 14℃-16℃) and artificial temperature control (18℃-19℃) for hatching. Artificial temperature control is achieved by placing a heating rod (brand: SenSen intelligent fish tank heating rod; model: URB-1000; temperature control range: 20-35℃; heating tube material: explosion-proof quartz tube) to make the hatching water temperature rise slowly by 1℃ every 24 hours until the water temperature is maintained at a constant temperature of 18℃-19℃ for incubation under artificial temperature control conditions such as circulating water aeration and sterilization. The fertilized eggs reach the middle gastrula after 54 hours, and begin to hatch after 119 hours. The hatching is completed after 153 hours, and the fry are 8.79-9.53mm long; after 217 hours, the fry are 9.62-10.45mm long and start to swim and feed. They are fed with Artemia nauplii 4 times a day. Every 10,000 fry are fed with 20g of nauplii hatched from Artemia eggs every day. Adjust the feeding amount according to the eating conditions of the fry; when the fry are about 3 cm long, put the fry in the homemade hatchery into a circulating breeding barrel with a diameter of 2-3m and feed them with high-protein artificial feed (high-end marine fish fry microparticle compound feed, particle size: S1, crude protein: greater than or equal to 52). Feed 4 times a day, and the feeding amount is 3% of the fry's body weight. Check the growth status of the fry under a microscope every morning. If parasites and other pests are found, use drugs to prevent and treat them in time. The following conclusions were drawn from comparative hatching experiments: natural spring water (water temperature: 14℃-16℃) was used for incubation of fertilized eggs, and the time for fertilized eggs to hatch was 216h-256h; artificial temperature control was used for incubation, and the time for fertilized eggs to hatch was 119h-153h; the growth rates of fry hatched from the same batch of fertilized eggs on the same day were compared after 29 days of cultivation: the fry hatched under spring water temperature of 14℃-16℃ grew to 13mm in length, and the fry hatched under artificial temperature control water temperature of 18℃-19℃ grew to 23mm in length. The growth rate of fry hatched under artificial temperature control conditions was more than twice that of fry cultivated under natural water temperature conditions.

[0044] As shown in Table 1 below, the present invention shows that the first generation of wild schizothorax roe obtained by artificial breeding can grow normally through artificial cultivation methods, and the second generation can be successfully bred after reaching sexual maturity. The incubation of wild schizothorax fertilized eggs is most ideal at 18°C-19°C, the water is neutral or slightly alkaline, and the water flow rate in the hatching bucket is 0.02-0.05m / s. Wild schizothorax matures late, usually 3-year-old males and 4-year-old females begin to mature and reproduce, with low fertility. Generally, the number of eggs carried by 4-year-old females is 7,000-8,000. Wild schizothorax spawns once, and the spawning time varies according to regional climate and environmental differences. Lugu Lake has a large temperature difference between day and night. Therefore, April to June each year is usually the peak period for spawning and reproduction of wild schizothorax, and it is also the best time for artificial breeding; after June, the ovaries gradually degenerate, and artificial breeding is no longer suitable. Similar to most schizothorax fish, the fertilized eggs of wild schizothorax are relatively large in shape, and can reach about 2mm after absorbing water. Therefore, the embryonic development time is relatively long. Under the condition of water temperature of 12℃-19.5℃, it takes more than 110 hours for fertilized eggs to develop into fry. During this period, many environmental factors can affect the hatching effect of fertilized eggs. If they are not properly controlled, it is very easy to cause large-scale death of fertilized eggs. Therefore, it is necessary to pay close attention to water temperature, water quality, water flow speed and other conditions to improve the hatching rate of fertilized eggs. Given that this research work was only completed in the Lugu Lake Basin of Jinsha River, whether the relevant technologies and methods summarized can be fully suitable for artificial breeding of this fish in other regions still needs further research.

[0045]

Claims

1. A method for artificial insemination of wild schizothorax schizothorax and hatching of fertilized eggs, characterized in that: The steps include: Step 1: Build broodstock domestication facilities: The domestication facilities use aquaculture barrels made of food-grade HDPP polypropylene, two large barrels, two medium barrels, and five temporary small barrels; each breeding barrel is independently equipped with a three-layer turnover box with a length of 60cm, a width of 40cm, and a height of 66cm as a water circulation trickle filter box water circulation device; Step 2: Collection and fishing of broodstock: In order to ensure the pure quality of the original species and the uniqueness of the broodstock traits, local fishermen with rich experience are invited to serve as the main catchers to carry out fishing operations. The fishing is carried out using a polyethylene three-layer gill net; Step 3: Water injection: Connect the water pipes to the breeding barrels to introduce mountain spring water, keep the water volume sufficient and fresh, and provide good environmental conditions for the breeding habitat. The domestication barrels are arranged in a cool and quiet room, with independent water inlet and outlet for each breeding barrel. The transparency is greater than 120cm, the dissolved oxygen is not less than 5mg / L, the pH value is 6.5-7.5, and the water temperature is 12.1-19.5℃; Step 4: Trauma prevention and disinfection of broodstock: smallmouthed schizothorax caught in the wild are placed in a temporary holding box on board: the volume is 80L, and a simple disinfection treatment is carried out with 4‰ salt and 10pp ginger solution for 1h-1.5h to reduce the aggravation of the fishing net hanging marks. After the captured wild broodstock are transported to the domestication point in the temporary holding box, the water temperature is adjusted to the same as the water temperature of the temporary holding small bucket, and then the fish body is smeared with gentian violet solution to disinfect the fish net hanging marks, and then placed in the temporary holding bucket for temporary holding; the next day, 450 liters of water are used for 48h of methylene blue 4 tablets and 10ppm of oxytetracycline hydrochloride for prevention and treatment, and the fish body hanging marks are treated with medicated bath every two days for 48h, and 3 times in a row to try to heal the fish body hanging marks; Step 5: Breeding and cultivation of broodstock: When broodstock are first caught, they have no appetite. After several days, the marks of gillnet on the broodstock and their physical condition recover. Try feeding them with thawed red nematodes. The results show that the broodstock actively eat. Then add artificial sturgeon slow-sinking expanded feed, particle size: 2.0, crude protein: greater than or equal to 41.0; Step 6: Artificial induction of spawning: After the captured smallmouthed schizothorax is transported to the taming site, the water temperature in the transport box is adjusted to be consistent with the water temperature in the temporary holding barrel. The water temperature in the taming barrel is maintained at 11-16°C. Then the broodstock are measured, weighed, and the sex is identified. The wounds of the broodstock are smeared with gentian violet solution and placed in a 500L temporary holding barrel for temporary fertilization. Unpolluted mountain spring water is introduced through a 20mm PVC pipe to continuously add new water to the temporary holding barrel, and oxygen is added with an aeration pump to fully stir the water in the barrel to stimulate the development of the broodstock's gonads. Increase the water in and out of the broodstock temporary holding barrel before inducing spawning. The water temperature in the temporary tank is changed every 20 to 25 minutes. A heating rod is placed in the temporary tank to gradually increase the water temperature by 1°C every 24 hours until the water temperature is maintained at a constant temperature of 19.5°C, with a slightly alkaline pH, 6.0 to 6.4 mg / L dissolved oxygen, 16 to 17 salinity, and ammonia nitrogen <0.1 mg / L. The water flow stimulation is continuously increased to make the male and female fish reach a similar stage of gonadal maturity. The sexual maturity of the broodstock is observed at any time. When the male and female fish are found chasing each other, artificial insemination is performed when the ratio of male to female reaches 1:

2. Step 7: Artificial insemination: dry insemination is adopted. First, the instruments are disinfected. The ratio of female fish to male fish is 1:2-7. The eggs and sperms of sexually mature male and female schizothorax are squeezed into the prepared vessels from their abdomens, and the two are stirred gently with chicken feathers for 30 seconds to fully mix them. After standing for 5 minutes, add appropriate amount of clean water to wash the fertilized eggs, preferably to submerge the sperm eggs, continue stirring for 20 seconds, and then wash them with the prepared clean water for 3 times to obtain fertilized eggs. The diameter of the fertilized eggs before absorbing water is 2.3-2.9mm, and the diameter of the eggs after absorbing water is 2.8-3.3mm. The fertilized eggs obtained are hatched in situ. Step 8: Artificial incubation: The fertilized eggs are placed in a pre-numbered homemade open cage in a bucket for incubation. The homemade open cage device is made of 30-40 mesh mesh with specifications of 54x400x17cm. The cage device used is soaked in 80ml of potassium permanganate solution per liter of water for 24 hours a few days in advance and then exposed to the sun for disinfection and sterilization before use. Each incubator is placed with 3000-4000 fertilized eggs. The water depth of the incubator is controlled at 15-20cm. During the incubation period, dead eggs are sucked out with a plastic straw at any time. The eggs and residues are kept in the incubator to keep the water fresh and the dissolved oxygen sufficient to prevent the occurrence of diseases and pests such as Saprolegniasis. The fertilized eggs are incubated with natural spring water at a water temperature of 14-16°C. The incubation and artificial temperature control are at 18-19°C for comparative experimental incubation. Artificial temperature control is achieved by placing a heating rod to control the incubation water temperature at 18-19°C for incubation under artificial temperature control conditions such as circulating water oxygenation and sterilization. The fertilized eggs reach the middle stage of the gastrula in 54 hours, and begin to hatch after 119 hours. The hatching is completed at 153 hours. The fry are 8.79-9 .53mm; after 217h, when the fry are 9.62-10.45mm long and start to swim and feed, feed Artemia nauplii 4 times a day, and feed 20g Artemia nauplii hatched from eggs per 10,000 fry every day; adjust the feeding amount according to the fry's eating situation; when the fry are about 3cm long, put the fry in the homemade hatching box into a circulating breeding barrel with a diameter of 2-3m and feed them with high-protein artificial feed, feed them 3 times a day, and the feeding amount is 3% of the fry's body weight. Check the fry's growth status under a microscope every morning. If parasites are found, Insects and other diseases and pests must be controlled in time with drugs; natural spring water is used for hatching fertilized eggs, the water temperature is 14℃-16℃, and the time for fertilized eggs to hatch is 216h-256h; artificial temperature control is used for hatching and the time for hatching is 119h-153h; the same batch of fry hatched on the same day are cultivated for 29 days. The fry hatched under natural water temperature of 14℃-16℃ grow to 13mm in length, and the fry hatched under artificial temperature control of 18℃-19℃ grow to 23mm in length, which is more than twice the growth rate of fry cultivated under natural water temperature conditions.

2. The method for artificial insemination and fertilized egg hatching of a wild Schizothorax schizothorax according to claim 1, characterized in that: The fishing net is a polyethylene three-layer barbed net, the net is woven with double knots and used horizontally, and the upper line of the net is equipped with foam plastic floats at certain intervals and the lower line is equipped with lead sinkers.

3. The method for artificial insemination and fertilized egg hatching of a wild Schizothorax microstomatis according to claim 1, characterized in that: The collection and capture of broodstock is to ensure the pure quality of the original species and the uniqueness of the broodstock traits.

4. The method for artificial insemination and fertilized egg hatching of wild Schizothorax microstomatis according to claim 1, characterized in that: During the artificial induction of spawning of broodstock, the captured small-mouthed schizothorax are transported to the taming site, and the water temperature in the transport box is adjusted to be consistent with the water temperature in the temporary holding barrel. The water temperature in the aquaculture barrel is maintained at 11-16°C. The broodstock are then measured, weighed, and the sex is identified. The wounds of the broodstock are smeared with gentian violet solution and placed in a 500L temporary holding barrel for temporary holding. Unpolluted mountain spring water is introduced through a 20mm PVC pipe, and new water is continuously added to the temporary holding barrel. Aeration is added with an aeration pump to fully stir the water in the barrel. To stimulate the development of broodstock gonads, increase the amount of water in and out of the broodstock temporary holding barrel before inducing spawning. Exchange the water in the temporary holding barrel every 20 to 25 minutes. Place a heating rod in the temporary holding barrel to slowly increase the water temperature by 1°C every 24 hours until the water temperature is maintained at a constant temperature of 19.5°C, with a weakly alkaline pH, 6.0 to 6.4 mg / L of dissolved oxygen, 16 to 17, and ammonia nitrogen <0.1 mg / L. Continuously increase water flow stimulation so that both male and female fish reach similar gonad maturity periods.

5. The method for artificial insemination and fertilized egg hatching of wild Schizothorax microstomatis according to claim 1, characterized in that: When the hatched fry start to swim and feed, they are fed with Artemia nauplii as training food. Artemia nauplii are fed 4 times a day. For every 10,000 fry, 20g of nauplii hatched from Artemia eggs are fed every day. The feeding amount is adjusted according to the eating habits of the fry. When the fry are about 3cm long, the fry in the homemade incubator are placed in a circulating breeding barrel with a diameter of 2-3m and fed with high-protein artificial feed.

6. The method for artificial insemination and fertilized egg hatching of wild Schizothorax microstomatis according to claim 1, characterized in that: The homemade open cage device is made of 30-40 mesh mesh with specifications of 54x400x17cm. The cage device is used after being soaked in 80ml of potassium permanganate solution per liter of water for 24 hours a few days in advance and then exposed to the sun for disinfection and sterilization before use. Each incubator box contains 3000-4000 fertilized eggs, and the water depth of the incubator box is controlled at 15-20cm.

Images