Seedling cultivation method for artificial propagation of light botia superba

Abstract

The invention discloses an offspring seed cultivation method for artificial propagation of light botia superba, and belongs to the technical field of aquaculture. The fry breeding method comprises the steps that doxycycline hydrochloride, MS222, multivitamins and ginger paste are added during parent fish transportation, and a two-stitch method is adopted for spawning induction during artificial spawning induction. The two-needle method comprises the following steps: firstly, injecting luteinizing hormone releasing hormone A2 and domperidone maleate into female fish; and secondly, injecting luteinizing hormone releasing hormone A2, domperidone maleate and chorionic gonadotropin into the female fish, and synchronously injecting luteinizing hormone releasing hormone A2 and domperidone maleate into the male fish. According to the method, the artificial propagation of the botia reevesii is realized, the induced spawning rate is greater than or equal to 90%, the fertilization rate is greater than or equal to 90%, the hatching rate is greater than or equal to 92%, the fry survival rate is greater than or equal to 88%, and the transportation death rate The method provides reliable technical support for resource protection, large-scale production, development and utilization of the botia delavayi.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, in particular to a seedling cultivation method for artificial propagation of light brown loach. Background Art

[0002] Nemacheilus subfuscus belongs to the order Cypriniformes, family Cobitidae, genus Nemacheilus. The body of the Nemacheilus subfuscus is elongated, slightly laterally flattened, and has a short caudal stalk. The head is conical, with the head height slightly smaller than the head width. The mouth is inferior. The lips are narrow, with shallow wrinkles on the lip surface, and a tooth-shaped protrusion located in the middle of the upper jaw. The distance from the base of the dorsal fin to the head is slightly greater than the distance from the base of the dorsal fin to the end of the caudal vertebrae; the end of the pelvic fin is far beyond the anus. The caudal fin is deeply concave. The anus is not close to the origin of the anal fin. The body is covered with small scales, and the scales are sparse in the area above the pectoral fin. The lateral line is thin and tubular, gradually becoming sparse towards the caudal fin, reaching or not reaching the base of the caudal fin. There are many brown transverse stripes on the back and sides of the body, with 5-9 transverse stripes in front of the dorsal fin and 4-5 transverse stripes behind the dorsal fin. A small, omnivorous fish, it often lives in gravel crevices and banks of tributaries. Its breeding season is mainly concentrated in March and April in spring, and its reproductive capacity is relatively low.

[0003] In recent years, human activities, overfishing, geological disasters, and other factors have significantly impacted the habitat of the brown loach, leading to a significant decline in its wild population and abundance, necessitating urgent conservation efforts. Research into artificial breeding techniques and the implementation of stock enhancement and release are key tools for protecting the brown loach population. However, current research on the brown loach is scarce, and existing technologies have not reported on its artificial propagation or seedling cultivation. Traditional methods are insufficient to meet resource conservation needs. Summary of the Invention

[0004] The present invention aims to provide a method for artificially propagating light brown loach seedlings to address the problems of the prior art. Examples of the present invention demonstrate a 90%-100% induced fertilization rate, a 90%-97% fertilization rate, a 92%-98% hatching rate, an 88%-96% seedling survival rate, and zero mortality during transportation.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a fry cultivation method for artificial propagation of light brown loach, comprising the steps of adding doxycycline hydrochloride, MS222, complex vitamins and ginger paste when transporting parent fish, and inducing labor by using a two-needle method during artificial induction of labor.

[0007] Furthermore, the two-injection method is: first, injecting luteinizing hormone-releasing hormone A2 and dioxigenin maleate into female fish; second, injecting luteinizing hormone-releasing hormone A2, dioxigenin maleate and chorionic gonadotropin into female fish, and simultaneously injecting luteinizing hormone-releasing hormone A2 and dioxigenin maleate into male fish.

[0008] Furthermore, the first needle and the second needle are separated by 12 hours.

[0009] Furthermore, the method includes the following steps: broodstock capture, broodstock transportation, broodstock breeding, artificial induction of spawning, artificial insemination, fertilized egg hatching, fry cultivation and seed cultivation.

[0010] The broodstock are harvested from March to April each year.

[0011] After the broodstock are caught, the method further comprises the step of selecting the broodstock: the light brown loach broodstock are classified, and those with obvious golden eggs observed on the abdomen are female mature broodstock, and those with milky white semen flowing out when the abdomen is lightly pressed are male mature broodstock.

[0012] Furthermore, during the artificial insemination, the ratio of the number of eggs to sperm is (1-3):(1-2).

[0013] Furthermore, in the artificial insemination, the sperm and the egg are mixed and then clean water is added to activate the sperm.

[0014] Furthermore, when the fertilized eggs are hatched, the laying density is 0.8-10,000 grains / m 2 , water temperature is 19-21℃, and water is exchanged 8-10 times / day.

[0015] During the larvae cultivation, no feeding is done for 0-3 days.

[0016] Furthermore, when the fry are cultivated, they are fed with Artemia at 4-8 days old, fed with a mixture of Artemia and Shengsuo S1 microcapsule material at 9-12 days old, and fed with Shengsuo S1 microcapsule material at 13-20 days old.

[0017] Furthermore, when the seedlings are cultivated, they are fed with Shengsuo S2 microcapsule material from 21 to 40 days old; they are fed with Shengsuo S3 microcapsule material from 41 to 70 days old; and they are fed with carp compound feed after 71 days.

[0018] The present invention also provides a method for improving the transportation survival rate of light brown loach broodstock, comprising the step of adding the doxycycline hydrochloride, MS222, compound vitamins and ginger paste during transportation of the light brown loach broodstock.

[0019] The present invention also provides a method for improving the artificial induction rate of light brown striped loach, comprising the step of using the two-needle method to induce labor during artificial induction of the light brown striped loach.

[0020] The present invention discloses the following technical effects:

[0021] The present invention addresses the pain points of the light brown loach, such as its short breeding period, high difficulty in inducing spawning, and low seedling survival rate. By innovating the harvesting, transportation, inducing spawning, and cultivation processes, it has achieved artificial large-scale breeding of the light brown loach for the first time, providing a key technological breakthrough for biodiversity conservation and sustainable utilization. Specifically, the present invention significantly improves the efficiency of artificial breeding of the light brown loach by optimizing the transportation formula and the combination of oxytocin. The embodiments of the present invention show that the induced spawning rate reaches 90%-100%, the fertilization rate is 90%-97%, the hatching rate is 92%-98%, the seedling survival rate is 88%-96%, and zero mortality is achieved during the transportation process. The breeding method provided by the present invention breaks through the problem of resource decline of the light brown loach due to its low fertility and limited habitat, fills the gap in artificial breeding technology of this species at home and abroad, provides an efficient technical solution for reproduction and release, and provides guidance for the artificial breeding, resource protection, large-scale production, and development and utilization of the light brown loach. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a picture of a mature male broodstock;

[0024] Figure 2 This is a picture of a female mature broodstock;

[0025] Figure 3 This is a picture of a fertilized egg;

[0026] Figure 4 This is a picture of newly hatched fry;

[0027] Figure 5 This is a picture of juvenile fish;

[0028] Figure 6 For pictures of juvenile fish;

[0029] Figure 7 This is a picture of an adult fish. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Doxycycline hydrochloride was purchased from Henan Zhongnong Kangxu Trading Co., Ltd., item number X3010036; MS222 was purchased from Fujian Jinjiang Aquatic Products Co., Ltd.; multivitamins were purchased from Hunan Kuai Da Bai Kang Feed Co., Ltd. Liuyang Branch; ginger paste was purchased from Yongji Bailing Baicao Biotechnology Co., Ltd.; luteinizing hormone-releasing hormone (LHRH) A2 was purchased from Ningbo Sansheng Biotechnology Co., Ltd., veterinary drug number 110912655; dioxinone maleate (DOM) was purchased from Ningbo Sansheng Biotechnology Co., Ltd. The company, veterinary drug ID number 110912944; human chorionic gonadotropin (HCG) was purchased from Ningbo Second Hormone Factory, veterinary drug ID number 110252207; carp compound feed was purchased from Tongwei Co., Ltd.; the Yushchenko incubator was purchased from Xiamen Chongrui Aquatic Technology Development Co., Ltd.; and artemia was purchased from Wudi Haiji Aquatic Products Co., Ltd. The differences between Shengsuo S1 microencapsulated feed, Shengsuo S2 microencapsulated feed, and Shengsuo S3 microencapsulated feed (all purchased from Shandong Shengsuo Feed Technology Co., Ltd.) lie in their suitable fry length, feed amount, and color. Shengsuo S1 microencapsulated feed is suitable for 6-10mm fry, with a dosage of 0.35g / 1,000 fry / time; Shengsuo S2 microencapsulated feed is suitable for 9-14mm fry, with a dosage of 0.7g / 1,000 fry / time; and Shengsuo S3 microencapsulated feed is suitable for 13-18mm fry, with a dosage of 1.5g / 1,000 fry / time.

[0036] Example 1

[0037] A method for cultivating artificially propagated seedlings of light brown loach

[0038] On the evening of March 26, 2024, at 18:00, two ground traps were set in the backwater near Didong Village on the lower reaches of the Yarlung Zangbo River. At 7:00 on March 27, 2024, the ground traps were retrieved and 28 light brown loach broodstock were obtained.

[0039] 28 light brown loach broodstock were classified, and those with obvious golden eggs observed in their abdomen were female mature broodstock ( Figure 1 ) 10 fish, milky white semen flows out when the abdomen is gently pressed, which is a mature male broodstock ( Figure 2 ) 12, and 6 other reserve broodstock;

[0040] Use 70cm straight oxygen bags to pack light brown loach. When packing, inject 1 / 3 of clear fresh river water into each bag. Add 0.1g doxycycline hydrochloride, 0.2g MS222, 9g multivitamins, and 1mL ginger paste into the water. Pack 3 bags in total. Put 12 female mature broodstock of light brown loach, 10 male mature broodstock, and 6 reserve broodstock into each bag. After filling with oxygen, tie the bags tightly and transport them to a fiberglass circular tank with a diameter of 1m and a depth of 80cm. Water enters the tank from the side and drains from the bottom. The water inlet and outlet are smooth and independent. The bottom of the tank is tilted toward the middle drain outlet. The water depth is controlled at 50cm. Put 2 20cm long PVC water pipe;

[0041] The packaged bag was placed in a culture tank and soaked for 30 minutes, then opened and slowly placed in the culture tank. Three packaged bags were placed in three tanks with three types of light brown loach broodstock; the mature broodstock were not fed, and the water exchange frequency was controlled to be 3 times / day; the reserve broodstock were fed once at 8:00 and 20:00 every day with a carp compound feed, with a feeding amount of 1.5 grams / time (not exceeding 2% of body weight). When feeding, the feed was crushed and kneaded into dough with water, and the leftover bait and feces were sucked out 1 hour after feeding. The water exchange frequency was controlled to be 3 times / day, and routine disinfection and care were carried out;

[0042] On March 27, 2024, at 12:00, artificial induction of spawning was performed on mature broodstock using a two-injection method. First, the female mature broodstock (obvious golden eggs could be seen on the abdomen) was injected with luteinizing hormone-releasing hormone (LHRH) A2 3μg / kg + dioxigenin maleate (DOM) 2mg / kg. 12 hours later, the female broodstock was injected with luteinizing hormone-releasing hormone (LHRH) A2 15μg / kg + dioxigenin maleate (DOM) 10mg / kg + human chorionic gonadotropin (HCG) 2000IU / kg. When the second injection of the female mature broodstock was given, the male mature broodstock (milky white semen flowed out when the lower abdomen was lightly pressed) was injected with luteinizing hormone-releasing hormone (LHRH) A2 5μg / kg + dioxigenin maleate (DOM) 2mg / kg.

[0043] The average water temperature was 20.0°C. 12 hours after the second injection, the female fish was first wiped dry, and the female abdomen was gently pressed from top to bottom to squeeze the mature eggs into a clean, dry plastic basin. The male fish was then wiped dry, and the male semen was squeezed out from the top to bottom. Subsequently, the semen was washed from the fish body onto the mature eggs with 0.9% NaCl solution; the male-to-female ratio was 10:12; the sperm and eggs were then evenly mixed with a feather, and clean water three times the volume of the sperm-egg mixture was added to activate the sperm. After stirring for 120 seconds, the excess water was poured out, and the fish were rinsed three times with clean water to remove mucus. After artificial insemination, the broodstock to be brooded were transferred to the reserve broodstock tank and raised in the same manner as the reserve broodstock. In this artificial breeding, 10 female light brown loaches were induced to spawn, 10 eggs were laid, 2000 mature eggs and 1800 fertilized eggs were obtained, with an induced spawning rate of 100% and a fertilization rate of 90%.

[0044] The fertilized egg ( Figure 3 ) Evenly spread on the Yushchenko incubator, with a density of 10,000 grains / m 2 , water exchange frequency 10 times / day, average water temperature 20.0℃, membrane rupture after 50h; the newly hatched fry obtained in this artificial breeding ( Figure 4 ) 1,700 tails, with a hatching rate of 94.44%.

[0045] After the larvae break the membrane, do not feed them for 0-3 days. Use a plastic straw to suck out dead eggs and fry every morning and evening. On the 4th day, transfer the larvae to a glass fiber reinforced plastic round tank with a diameter of 1m and a depth of 80cm. The water is fed from the side and drained from the bottom. The water inlet and outlet are smooth and independent. The bottom of the tank is tilted towards the middle drain outlet. The water depth is controlled at 50cm. Place 3 rods at the bottom of the tank. 1700 light brown loach larvae were stocked in a 20 cm long PVC water pipe. From 4 to 8 days, they were overfed with Artemia (2-3 g / time) every 4 hours. From 9 to 12 days, they were overfed with a mixture of Artemia and Shengsuo S1 microcapsule at a ratio of 1:1 (4 g / time in total). Figure 5 ); feed the fish with 5g of S1 microcapsule every 8 hours for 13-20 days; remove the remaining bait and feces 30 minutes after each feeding, control the water exchange frequency to 3 times per day, and perform routine disinfection and care; the artificial breeding of light brown loach juveniles ( Figure 6 ) More than 1,500 fish were found, with a seedling survival rate of 88.24%.

[0046] On day 21, the density of juvenile fish was diluted to 80 per tank. From day 21 to day 40, the juvenile fish were fed with Shengsuo S2 microcapsule feed once at 8:00 and 20:00 each day. From day 41 to day 70, the juvenile fish were fed with Shengsuo S3 microcapsule feed once at 8:00 and 20:00 each day. After day 71, the juvenile fish were fed with carp compound feed once at 8:00 and 20:00 each day. The feed was crushed and kneaded into dough before feeding. The feeding amounts of Shengsuo S2 microcapsule feed, Shengsuo S3 microcapsule feed and carp compound feed were 7 g / time (with water), 10 g / time (with water) and 15 g / time (with water) respectively. During the rearing period, the residual bait and feces were sucked out 1 hour after each feeding. The water exchange frequency was controlled to 3 times / day. Routine disinfection and care were performed. After day 70, the adult fish ( Figure 7 ).

[0047] Example 2

[0048] A method for cultivating artificially propagated seedlings of light brown loach

[0049] On the evening of April 8, 2024, at 7:00 PM, two ground traps were set near Didong Village on the lower reaches of the Yarlung Zangbo River and in the backwaters of the tributaries Ximo River and Lingongri River. At 8:00 AM on April 9, 2024, the ground traps were retrieved and 85 broodstock of light brown loach were obtained.

[0050] 85 light brown loach broodstock were classified. Among them, 40 were female mature broodstock with obvious golden eggs observed in their abdomen, 36 were male mature broodstock with milky white semen flowing out when their abdomen was gently pressed, and 9 other reserve broodstock were found.

[0051] Use 70cm straight oxygen bags to pack light brown loach. When packing, inject 1 / 3 of clear fresh river water into each bag. Add 0.1g doxycycline hydrochloride, 0.3g MS222, 10g multivitamins, and 1.5mL ginger paste into the water. Pack 5 bags in total. Put 20 female mature broodstock of light brown loach, 20 female mature broodstock of light brown loach, 18 male mature broodstock, 18 male mature broodstock and 9 reserve broodstock into each bag. After filling with oxygen, tie the bag tightly and transport it to a fiberglass circular tank with a diameter of 1m and a depth of 80cm. Water enters the tank from the side and drains from the bottom. The water inlet and outlet are smooth and independent. The bottom of the pool is tilted toward the middle drain outlet. The water depth is controlled at 60cm. Put 2 at the bottom of the tank. 30cm long PVC water pipe;

[0052] The packaged bags were placed in a culture tank and soaked for 30 minutes, then opened and slowly placed in the culture tank. Five packaged bags were divided into three tanks and three types of light brown loach broodstock were placed; the mature broodstock were not fed, and the water exchange frequency was controlled to be 3 times / day; the reserve broodstock were fed once at 8:00 and 20:00 every day with a carp compound feed, with a feeding amount of 3.5 grams / time (not exceeding 2% of body weight). When feeding, the feed was crushed and kneaded into dough with water, and the leftover bait and feces were sucked out 1 hour after feeding. The water exchange frequency was controlled to be 3 times / day, and routine disinfection and care were carried out;

[0053] At 12:00 on April 9, 2024, artificial induction of spawning was performed on mature broodstock using a two-injection method. First, the female mature broodstock (obvious golden eggs can be seen on the abdomen) was injected with luteinizing hormone-releasing hormone (LHRH) A2 4μg / kg + dioxigenin maleate (DOM) 2mg / kg. 12 hours later, the female broodstock was injected with luteinizing hormone-releasing hormone (LHRH) A2 15μg / kg + dioxigenin maleate (DOM) 9mg / kg + human chorionic gonadotropin (HCG) 2000IU / kg. When the second injection of the female mature broodstock was given, the male mature broodstock (milky white semen flowed out when the lower abdomen was lightly pressed) was injected with luteinizing hormone-releasing hormone (LHRH) A2 5μg / kg + dioxigenin maleate (DOM) 3mg / kg.

[0054] The average water temperature was 20.0°C. 11 hours after the second injection, the female fish were first wiped dry, and the female abdomen was gently pressed from top to bottom to squeeze the mature eggs into a clean, dry plastic basin. The male fish were then wiped dry, and the male semen was squeezed from the top to bottom. The semen was then washed from the fish body onto the mature eggs with 0.9% NaCl solution. The male-to-female ratio was 36:36. The sperm and eggs were then mixed evenly with a feather, and clean water five times the volume of the sperm-egg mixture was added to activate the sperm. After stirring for 120 seconds, the excess water was poured out, and the fish were rinsed three times with clean water to remove mucus. After artificial insemination, the broodstock were transferred to the reserve broodstock tank and reared in the same manner as the reserve broodstock. In this artificial breeding, 36 female light brown loaches were induced to spawn, 34 of which laid eggs, resulting in 7,000 mature eggs and 6,500 fertilized eggs, with an induced spawning rate of 94.44% and a fertilization rate of 92.86%.

[0055] Spread the fertilized eggs evenly on the Yushchenko incubator at a density of 10,000 eggs / m 2 The water was exchanged 10 times per day, the average water temperature was 20.0℃, and the membranes were broken after 50 hours. 6,200 newly hatched fry were obtained in this artificial breeding, with a hatching rate of 95.38%.

[0056] After the larvae break the membrane, do not feed them for 0-3 days. Use a plastic straw to suck out dead eggs and fry every morning and evening. On the 4th day, transfer the larvae to a glass fiber reinforced plastic round tank with a diameter of 1m and a depth of 80cm. The water is fed from the side and drained from the bottom. The water inlet and outlet are smooth and independent. The bottom of the tank is tilted towards the middle drain outlet. The water depth is controlled at 50cm. Place 3 rods at the bottom of the tank. 20 cm long PVC water pipes were used to stock 2,000 light brown striped loach fry in each tank; from 4 to 8 days, they were overfed with Artemia (8-12 g / time) every 4 hours; from 9 to 12 days, they were overfed with a mixture of Artemia and Shengsuo S1 microcapsule feed at a mixing ratio of 1:1 (20 g / time in total) every 8 hours; from 13 to 20 days, they were overfed with Shengsuo S1 microcapsule feed (25 g / time) every 8 hours; after each feeding during the cultivation period, the remaining bait and feces were sucked out 30 minutes after the completion of each feeding, the water exchange frequency was controlled at 3 times / day, and routine disinfection and care were performed; 6,000 light brown striped loach fry were obtained through artificial breeding, with a seedling survival rate of 96.77%.

[0057] On the 21st day, the density of juvenile fish was diluted to 100 fish / tank, and Shengsuo S2 microcapsule was fed once a day at 8:00 and 20:00 from the 21st day to the 40th day; Shengsuo S3 microcapsule was fed once a day at 8:00 and 20:00 from the 41st day to the 70th day; after the 71st day, carp compound feed was fed once a day at 8:00 and 20:00, and the feed was crushed and kneaded into dough before feeding; the feeding amounts of Shengsuo S2 microcapsule, Shengsuo S3 microcapsule and carp compound feed were 30 g / time (with water), 50 g / time (with water) and 70 g / time (with water), respectively; during the cultivation period, the remaining bait and feces were sucked out 1 hour after each feeding, the water exchange frequency was controlled to 3 times / day, and routine disinfection and care were carried out.

[0058] Example 3

[0059] A method for cultivating artificially propagated seedlings of light brown loach

[0060] On the evening of April 15, 2024, at 18:00, two ground traps were set near Didong Village on the main stream of the lower reaches of the Yarlung Zangbo River and in the backwaters of the tributaries of the Deergong River and the Lingongri River. At 8:00 on April 16, 2024, the ground traps were retrieved and 90 light brown loach broodstock were obtained;

[0061] 90 light brown loach broodstock were classified. Among them, 38 were female mature broodstock with obvious golden eggs observed in their abdomen, 40 were male mature broodstock with milky white semen flowing out when their abdomen was lightly pressed, and 12 were other reserve broodstock.

[0062] Use 70cm straight oxygen bags to pack light brown loach. When packing, inject 1 / 4 of clear fresh river water into each bag. Add 0.2g doxycycline hydrochloride, 0.4g MS222, 8g multivitamins, and 2mL ginger paste into the water. Pack 5 bags in total. Put 20 female mature broodstock of light brown loach, 18 female mature broodstock of light brown loach, 20 male mature broodstock, 20 male mature broodstock, and 12 reserve broodstock into each bag. After filling with oxygen, tie the bag tightly and transport it to a fiberglass circular tank with a diameter of 1m and a depth of 80cm. Water enters the tank from the side and drains from the bottom. The water inlet and outlet are smooth and independent. The bottom of the pool is tilted toward the middle drain outlet. The water depth is controlled at 60cm. Put 3 30cm long PVC water pipe;

[0063] The packaged bags were placed in a culture tank and soaked for 30 minutes, then opened and slowly placed in the culture tank. Five packaged bags were divided into three tanks and three types of light brown loach broodstock were placed; the mature broodstock were not fed, and the water exchange frequency was controlled to be 3 times / day; the reserve broodstock were fed once at 8:00 and 20:00 every day with a carp compound feed, with a feeding amount of 7 grams / time (no more than 2% of body weight). When feeding, the feed was crushed and kneaded into dough with water, and the leftover bait and feces were sucked out 1 hour after feeding. The water exchange frequency was controlled to be 3 times / day, and routine disinfection and care were carried out;

[0064] At 12:00 on April 16, 2024, artificial induction of spawning was performed on mature broodstock using a two-injection method. First, the female mature broodstock (obvious golden eggs can be seen on the abdomen) were injected with luteinizing hormone-releasing hormone (LHRH) A2 3μg / kg + dioxigenin maleate (DOM) 3mg / kg. 12 hours later, the female broodstock were injected with luteinizing hormone-releasing hormone (LHRH) A2 14μg / kg + dioxigenin maleate (DOM) 9mg / kg + human chorionic gonadotropin (HCG) 2500IU / kg. When the female mature broodstock were injected with the second injection, the male mature broodstock (milky white semen flowed out when the lower abdomen was lightly pressed) were injected with luteinizing hormone-releasing hormone (LHRH) A2 6μg / kg + dioxigenin maleate (DOM) 3mg / kg.

[0065] The average water temperature was 21.0°C. Ten hours after the second injection, the female fish were first wiped dry, and the female abdomen was gently pressed from top to bottom to squeeze the mature eggs into a clean, dry plastic basin. The male fish were then wiped dry, and the male semen was squeezed from the top to bottom. The semen was then washed from the fish body onto the mature eggs with 0.9% NaCl solution. The male-to-female ratio was 38:40. The sperm and eggs were then mixed evenly with a feather, and clean water 5 times the volume of the sperm-egg mixture was added to activate the sperm. After stirring for 100 seconds, the excess water was poured out, and the fish were rinsed twice with clean water to remove mucus. After artificial insemination, the broodstock were transferred to the reserve broodstock tank and raised in the same manner as the reserve broodstock. In this artificial breeding, 38 female light brown loaches were induced to spawn, 35 of which laid eggs, resulting in 7,200 mature eggs and 7,000 fertilized eggs, with an induced spawning rate of 92.11% and a fertilization rate of 97.22%.

[0066] The fertilized eggs were evenly spread on the Yushchenko incubator at a density of 8,000 eggs / m 2 The water was exchanged 8 times per day, the average water temperature was 21.0℃, and the membranes were broken after 49 hours. 6,800 newly hatched fry were obtained in this artificial breeding, with a hatching rate of 97.14%.

[0067] After the larvae break the membrane, do not feed them for 0-3 days. Use a plastic straw to suck out dead eggs and fry every morning and evening. On the 4th day, transfer the larvae to a glass fiber reinforced plastic round tank with a diameter of 1m and a depth of 80cm. The water is fed from the side and drained from the bottom. The water inlet and outlet are smooth and independent. The bottom of the tank is tilted towards the middle drain outlet. The water depth is controlled at 50cm. Place 3 rods at the bottom of the tank. 30 cm long PVC water pipes were used to stock 3,000 light brown striped loach fry in each tank; from 4 to 8 days, they were overfed with Artemia (8-12 g / time) every 4 hours; from 9 to 12 days, they were overfed with a mixture of Artemia and Shengsuo S1 microcapsule feed at a mixing ratio of 1:1 (20 g / time in total) every 8 hours; from 13 to 20 days, they were overfed with Shengsuo S1 microcapsule feed (25 g / time) every 8 hours; after each feeding during the cultivation period, the remaining bait and feces were sucked out 30 minutes after each feeding, the water exchange frequency was controlled to 3 times / day, and routine disinfection and care were performed; 6,500 light brown striped loach fry were obtained in this artificial breeding, with a seedling survival rate of 95.59%.

[0068] On the 21st day, the density of juvenile fish was diluted to 80 fish / tank. From the 21st day to the 40th day, the juvenile fish were fed with Shengsuo S2 microcapsule feed once at 8:00 and 20:00 every day; from the 41st day to the 70th day, the juvenile fish were fed with Shengsuo S3 microcapsule feed once at 8:00 and 20:00 every day; after the 71st day, the carp compound feed was fed once at 8:00 and 20:00 every day. When feeding, the feed was crushed and watered into dough; the feeding amounts of Shengsuo S2 microcapsule feed, Shengsuo S3 microcapsule feed and carp compound feed were 30 grams / time (with water), 50 grams / time (with water) and 70 grams / time (with water), respectively; during the cultivation period, the remaining bait and feces were sucked out 1 hour after each feeding, the water exchange frequency was controlled to 3 times / day, and routine disinfection and care were carried out.

[0069] Example 4

[0070] A method for cultivating artificially propagated seedlings of light brown loach

[0071] On March 10, 2025, 30 female mature broodstock with obvious golden eggs observed on their abdomens were selected from the reserve broodstock cultured in Examples 1-3 and the light brown loaches collected from the backwaters near Didong Village in the lower reaches of the Yarlung Zangbo River in 2025. 30 male mature broodstock were selected, and milky white semen flowed out when the abdomen was lightly pressed.

[0072] On March 10, 2025, at 12:00, a two-injection method was used to induce spawning. First, the female mature broodstock were injected with luteinizing hormone-releasing hormone (LHRH) A2 4μg / kg + dioxigenin maleate (DOM) 2mg / kg. 12 hours later, the female broodstock were injected with luteinizing hormone-releasing hormone (LHRH) A2 15μg / kg + dioxigenin maleate (DOM) 10mg / kg + human chorionic gonadotropin (HCG) 2500IU / kg. When the female mature broodstock were injected with the second injection, the male mature broodstock (milky white semen flowed out when the lower abdomen was lightly pressed) were injected with luteinizing hormone-releasing hormone (LHRH) A2 6μg / kg + dioxigenin maleate (DOM) 3mg / kg.

[0073] The average water temperature was 19.0°C. 12 hours after the second injection, the female fish were first wiped dry, and the female abdomen was gently pressed from top to bottom to squeeze the mature eggs into a clean, dry plastic basin. The male fish were then wiped dry, and the male semen was squeezed out from the top to bottom. The semen was then rinsed from the fish body onto the mature eggs with 0.9% NaCl solution; the male-to-female ratio was 30:30. The sperm and eggs were then mixed evenly with a feather, and clean water four times the volume of the sperm-egg mixture was added to activate the sperm. After stirring for 120 seconds, the excess water was poured out, and the fish were rinsed three times with clean water to remove mucus. After artificial insemination, the broodstock were transferred to the reserve broodstock tank and raised in the same manner as the reserve broodstock. In this artificial breeding, 30 female light brown loaches were induced to spawn, 29 of which laid eggs, 5,500 mature eggs and 5,000 fertilized eggs were obtained, with an induced spawning rate of 96.67% and a fertilization rate of 90.90%.

[0074] Spread the fertilized eggs evenly on the Yushchenko incubator at a density of 10,000 eggs / m 2 The water was exchanged 8 times per day, the average water temperature was 19.0℃, and the membranes were broken after 51 hours. 4,900 newly hatched fry were obtained in this artificial breeding, with a hatching rate of 98.00%.

[0075] After the larvae break the membrane, do not feed them for 0-3 days. Use a plastic straw to suck out dead eggs and fry every morning and evening. On the 4th day, transfer the larvae to a glass fiber reinforced plastic round tank with a diameter of 1m and a depth of 80cm. The water is fed from the side and drained from the bottom. The water inlet and outlet are smooth and independent. The bottom of the tank is tilted towards the middle drain outlet. The water depth is controlled at 50cm. Place 3 rods at the bottom of the tank. 30 cm long PVC water pipes were used to stock 3,000 light brown striped loach fry in each tank; from 4 to 8 days, they were overfed with Artemia (6 to 10 g / time) every 4 hours; from 9 to 12 days, they were overfed with a mixture of Artemia and Shengsuo S1 microcapsule feed at a mixing ratio of 1:1 (a total of 15 g / time) every 8 hours; from 13 to 20 days, they were overfed with Shengsuo S1 microcapsule feed (20 g / time) every 8 hours; after each feeding during the cultivation period, the remaining bait and feces were sucked out 30 minutes after each feeding, the water exchange frequency was controlled to 3 times / day, and routine disinfection and care were performed; 4,500 light brown striped loach fry were obtained in this artificial breeding, with a seedling survival rate of 91.84%.

[0076] On the 21st day, the density of juvenile fish was diluted to 100 fish / tank. After the 21st day, the juvenile fish were fed with Shengsuo S2 microcapsule feed once at 8:00 and 20:00 every day; from 41 to 70 days, the juvenile fish were fed with Shengsuo S3 microcapsule feed once at 8:00 and 20:00 every day; after the 71st day, the juvenile fish were fed with carp compound feed once at 8:00 and 20:00 every day. When feeding, the feed was crushed and watered to form dough. The feeding amounts of Shengsuo S2 microcapsule feed, Shengsuo S3 microcapsule feed and carp compound feed were 25 g / time (with water), 40 g / time (with water) and 50 g / time (with water), respectively. During the cultivation period, the remaining bait and feces were sucked out 1 hour after each feeding, the water exchange frequency was controlled to 3 times / day, and routine disinfection and care were carried out.

[0077] Example 5

[0078] A method for cultivating artificially propagated seedlings of light brown loach

[0079] On March 15, 2025, 20 female mature broodstock with obvious golden eggs observed in the abdomen were selected from the reserve broodstock cultured in Examples 1-3 and the light brown loaches collected from the backwater near Didong Village in the lower reaches of the Yarlung Zangbo River in 2025. 20 male mature broodstock with milky white semen flowing out when the abdomen was lightly pressed were used for artificial breeding;

[0080] On March 15, 2025, at 12:00, a two-injection method was used to induce spawning. First, the female mature broodstock were injected with luteinizing hormone-releasing hormone (LHRH) A2 3μg / kg + dioxigenin maleate (DOM) 3mg / kg. 12 hours later, the female broodstock were injected with luteinizing hormone-releasing hormone (LHRH) A2 15μg / kg + dioxigenin maleate (DOM) 10mg / kg + human chorionic gonadotropin (HCG) 2000IU / kg. When the female mature broodstock were injected with the second injection, the male mature broodstock (milky white semen flowed out when the lower abdomen was lightly pressed) were injected with luteinizing hormone-releasing hormone (LHRH) A2 5μg / kg + dioxigenin maleate (DOM) 2mg / kg.

[0081] The average water temperature was 20.0°C. Eleven hours after the second injection, the female fish were first wiped dry, and the female abdomen was gently pressed from top to bottom to squeeze the mature eggs into a clean, dry plastic basin. The male fish were then wiped dry, and the male semen was squeezed from the top to bottom. Subsequently, the semen was washed from the fish body onto the mature eggs using 0.9% NaCl solution. The male-to-female ratio was 20:20. The sperm and eggs were then mixed evenly with a feather, and clean water four times the volume of the sperm-egg mixture was added to activate the sperm. After stirring for 120 seconds, the excess water was poured out, and the fish were rinsed three times with clean water to remove mucus. After artificial insemination, the broodstock were transferred to the reserve broodstock tank and reared in the same manner as the reserve broodstock. Twenty female light brown loaches were artificially bred and spawned, resulting in 4,000 mature eggs and 3,800 fertilized eggs, a 100% induced spawning rate, and a 95.00% fertilization rate.

[0082] Spread the fertilized eggs evenly on the Yushchenko incubator at a density of 10,000 eggs / m 2 The water was exchanged 8 times per day, the average water temperature was 19.0℃, and the membranes were broken after 51 hours. 3,500 newly hatched fry were obtained in this artificial breeding, with a hatching rate of 92.11%.

[0083] After the larvae break the membrane, do not feed them for 0-3 days. Use a plastic straw to suck out dead eggs and fry every morning and evening. On the 4th day, transfer the larvae to a glass fiber reinforced plastic round tank with a diameter of 1m and a depth of 80cm. The water is fed from the side and drained from the bottom. The water inlet and outlet are smooth and independent. The bottom of the tank is tilted towards the middle drain outlet. The water depth is controlled at 50cm. Place 3 rods at the bottom of the tank. 30 cm long PVC water pipes were used to stock 3000 light brown striped loach fry in each tank; from 4 to 8 days, they were overfed with Artemia (5-7 g / time) every 4 hours; from 9 to 12 days, they were overfed with a mixture of Artemia and Shengsuo S1 microcapsule feed at a mixing ratio of 1:1 (a total of 12 g / time) every 8 hours; after 13 days, they were overfed with Shengsuo S1 microcapsule feed (15 g / time) every 8 hours; after each feeding during the cultivation period, the remaining bait and feces were sucked out 30 minutes after the completion of each feeding, the water exchange frequency was controlled to 3 times / day, and routine disinfection and care were carried out; 3200 light brown striped loach fry were obtained in this artificial breeding, with a seedling survival rate of 91.43%.

[0084] On the 21st day, the density of juvenile fish was diluted to 100 fish / tank. After the 21st day, the juvenile fish were fed with Shengsuo S2 microcapsule feed once at 8:00 and 20:00 every day; from 41 to 70 days, the juvenile fish were fed with Shengsuo S3 microcapsule feed once at 8:00 and 20:00 every day; after the 71st day, the juvenile fish were fed with carp compound feed once at 8:00 and 20:00 every day. When feeding, the feed was crushed and watered to form dough. The feeding amounts of Shengsuo S2 microcapsule feed, Shengsuo S3 microcapsule feed and carp compound feed were 20 g / time (with water), 30 g / time (with water) and 40 g / time (with water), respectively. During the cultivation period, the remaining bait and feces were sucked out 1 hour after each feeding, the water exchange frequency was controlled to 3 times / day, and routine disinfection and care were carried out.

[0085] Comparative Example 1

[0086] On March 10, 2025, 10 female mature broodstock with obvious golden eggs observed on their abdomens were selected from the reserve broodstock cultured in Examples 1-3 and the light brown loaches collected from the backwaters near Didong Village in the lower reaches of the Yarlung Zangbo River in 2025. 12 male mature broodstock were selected, and milky white semen flowed out when the abdomen was lightly pressed.

[0087] The difference between the breeding method of this comparative example and that of Example 1 is that, during the transportation of the broodstock, the ingredients added in this comparative example are 3g salt, 0.5g MS222, 1g povidone iodine, and 1g allicin. The remaining steps are the same as those of Example 1.

[0088] Comparative Example 2

[0089] On March 10, 2025, 10 female mature broodstock with obvious golden eggs observed on their abdomens were selected from the reserve broodstock cultured in Examples 1-3 and the light brown loaches collected from the backwaters near Didong Village in the lower reaches of the Yarlung Zangbo River in 2025. 12 male mature broodstock were selected, and milky white semen flowed out when the abdomen was lightly pressed.

[0090] The difference between the breeding method of this comparative example and that of Example 1 is that, in this comparative example, when artificially inducing spawning of broodstock, the method adopted is a single-needle injection method. Female mature broodstock (obvious golden eggs can be seen on the abdomen) are injected with luteinizing hormone-releasing hormone (LHRH) A2 10 μg / kg + dioctyl maleate (DOM) 8 mg / kg + human chorionic gonadotropin (HCG) 2000 IU / kg, and male mature broodstock (milky white semen flows out when the lower abdomen is gently pressed) are injected with luteinizing hormone-releasing hormone (LHRH) A2 5 g / kg + dioctyl maleate (DOM) 4 mg / kg. The remaining steps are the same as in Example 1.

[0091] The results of artificial propagation of light brown loach seedlings in Examples 1-5 and Comparative Examples 1-2 are shown in Table 1.

[0092] Table 1

[0093]

[0094]

[0095] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for artificially propagating light brown loach seedlings, characterized in that: The method includes adding doxycycline hydrochloride, MS222, multivitamins and ginger paste when transporting broodstock, and using a two-injection method for artificial induction of labor.

2. The seedling cultivation method according to claim 1, wherein The two-injection method is as follows: first, injecting luteinizing hormone-releasing hormone A2 and dioxigenin maleate into female fish; second, injecting luteinizing hormone-releasing hormone A2, dioxigenin maleate and chorionic gonadotropin into female fish, and simultaneously injecting luteinizing hormone-releasing hormone A2 and dioxigenin maleate into male fish.

3. The seedling cultivation method according to claim 2, characterized in that: The first needle and the second needle are separated by 12 hours.

4. The seedling cultivation method according to claim 1, wherein The following steps are involved: Broodstock capture, broodstock transportation, broodstock farming, artificial induction of spawning, artificial insemination, hatching of fertilized eggs, fry cultivation and seed cultivation.

5. The seedling cultivation method according to claim 4, characterized in that: During the artificial insemination, the ratio of the number of eggs to the number of sperm is (1-3):(1-2).

6. The seedling cultivation method according to claim 4, characterized in that: When the fertilized eggs are hatched, the laying density is 0.8-10,000 grains / m 2 , water temperature is 19-21℃, and water is exchanged 8-10 times / day.

7. The seedling cultivation method according to claim 4, characterized in that: When the fry are cultured, they are fed with Artemia spp. at 4-8 days old, fed with a mixture of Artemia spp. and Sinso S1 microcapsule material at 9-12 days old, and fed with Sinso S1 microcapsule material at 13-20 days old.

8. The seedling cultivation method according to claim 4, characterized in that: When the seedlings are cultivated, the seedlings are fed with Shengsuo S2 microcapsule material at 21-40 days old; the seedlings are fed with Shengsuo S3 microcapsule material at 41-70 days old; and the seedlings are fed with carp compound feed after 71 days.

9. A method for improving the transport survival rate of light brown loach broodstock, characterized in that: The method comprises the steps of adding the doxycycline hydrochloride, MS222, multivitamins and ginger paste as claimed in claim 1 during transportation of the light brown loach broodstock.

10. A method for improving the artificial induced labor rate of light brown loach, characterized in that: The method comprises the step of using the two-needle method described in claim 1 to induce labor when artificially inducing labor in the light brown loach.

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