A method for artificial breeding of tropical sea cucumbers

Abstract

The present invention discloses a method for artificial breeding of tropical sea cucumbers. The method comprises the collection of sea cucumber parents, acquisition of gametes, fertilization and hatching, cultivation of planktonic larvae, attachment and cultivation of larvae, and intermediate culture of tropical sea cucumbers. For the first time, sea cucumbers of the jade foot, sea cucumbers of the brown ring, and sea cucumbers of the mikkelsen are bred by dissecting and obtaining sperm and eggs. The method provides a technical guarantee for the continuation and resource recovery of tropical sea cucumber populations that are on the verge of extinction and cannot undergo artificial induction of spawning. The method is simple and easy to operate for artificial breeding of tropical sea cucumbers, and can effectively overcome a series of problems in the seedling cultivation process, such as the inability to induce spawning / poor induction of spawning effect, unstable output, and unscientific feeding of baits. The success rate of artificial breeding of tropical sea cucumber seedlings is improved. The method does not damage key structures such as the body wall and other organs of the parent organism, and has a very broad application space in the field of artificial breeding and aquaculture of tropical sea cucumbers.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture, and more specifically, relates to a method for artificially breeding tropical sea cucumbers. Background Art

[0002] Tropical sea cucumbers primarily inhabit tropical and subtropical waters and have unique habits that differ from those of their northern cousins, the sea cucumber. Many species of tropical sea cucumbers are rich in natural active substances such as polysaccharides and saponins, which possess antioxidant, antibacterial, anti-inflammatory, and anti-tumor properties, greatly increasing their potential economic value. Tropical sea cucumbers also play an important role in submarine ecosystems. Through physiological activities such as feeding and excretion, they can reduce the organic matter content of sediments and increase the nutrient flux and dissolved oxygen exchange rate at the sediment-water interface. Furthermore, co-culturing tropical sea cucumbers with other farmed animals, such as fish and shrimp, can effectively regulate the aquaculture environment and improve the growth and survival rates of farmed animals. Therefore, tropical sea cucumbers can be used as biofilters in the construction of complex ecological aquaculture systems.

[0003] With rising consumer spending, global sea cucumber production has increased rapidly over the past few decades. However, under pressures from overfishing, environmental pollution, and climate change, natural sea cucumber stocks have declined rapidly in recent years, with some populations even nearing extinction. Research indicates that artificial breeding and stocking sea cucumbers are effective solutions to replenishing depleted wild populations.

[0004] Currently, one of the key approaches to restoring sea cucumber resources both domestically and internationally is stocking, which requires a stable supply of seedlings. Compared to commercially viable species like the imitation sea cucumber and the rough sea cucumber, artificial propagation techniques for most tropical sea cucumber species are still immature. For example, artificial induction methods for tropical sea cucumbers like the Tuwen white sea cucumber, the plum-flowered sea cucumber, the brown-ringed sea cucumber, and the Mieke sea cucumber are currently unavailable. During the artificial propagation of sea cucumbers, factors such as water quality, feed, predators, and management during the seedling rearing process can affect the growth, development, and survival of planktonic larvae. Many tropical sea cucumber species, such as the jade-foot sea cucumber, have a planktonic larval stage that is more than twice that of the imitation sea cucumber and the rough sea cucumber. In this lengthy process, even the slightest carelessness can lead to delayed seedling development or even mass mortality. Furthermore, sea cucumber seedling rearing has a significantly higher success rate when managed by experienced technicians than by less experienced ones. This is primarily due to the latter's tendency to rely on a rote understanding of key processes such as water quality control, feed preparation and formulation, and predator control, lacking practical experience.

[0005] Of the existing sea cucumber seedling techniques, only those for Stichopus imitatingus and Holothuria sclerotium are relatively mature, while artificial breeding technology for other tropical sea cucumber species is still in its infancy. It is well known that each sea cucumber species has its own unique biological characteristics. If the artificial breeding of other sea cucumber species is managed by completely imitating the seedling breeding manuals of species such as Stichopus imitatingus and Holothuria sclerotium, the results are often unsatisfactory. Therefore, the key links in the artificial breeding process of tropical sea cucumbers, including gamete acquisition, water quality control, unicellular bait algae cultivation, pest control, and larval rearing, must be standardized. From a practical perspective, while ensuring the effectiveness of seedling cultivation, the steps should be simplified as much as possible. This can avoid detours and significantly reduce the workload of seedling technicians, effectively reducing risks and increasing the success rate of seedling cultivation.

[0006] It is well known that artificial induction of labor is the most effective method for artificially breeding sea cucumbers by obtaining fertilized eggs. Generally speaking, methods of artificial induction include drying in the shade, water stimulation, temperature stimulation, and drug stimulation. However, in actual operations, we have found that although the gonads of some tropical sea cucumber species have matured, no matter what traditional methods are used under indoor conditions, they cannot naturally release sperm and eggs. Many sea cucumber species that are difficult to artificially induce labor, such as Tuwen Baini Sea Cucumber and Meihua Sea Cucumber, are already endangered in the wild, and their populations are no longer sufficient to support natural reproduction. Therefore, effective means must be taken to rescue and increase their number. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for artificially breeding tropical sea cucumbers, which can provide a technical guarantee for the continuation and resource recovery of tropical sea cucumber populations that are on the verge of extinction and cannot be artificially induced to produce.

[0008] A method for artificially breeding tropical sea cucumbers of the present invention comprises the following steps:

[0009] A. Water quality control: Take natural seawater and treat it until the pollutant content is lower than the second-class seawater quality standard;

[0010] B. Collection of sea cucumber parents: Collect tropical sea cucumbers with mature gonads;

[0011] C. Gamete acquisition: After emptying the intestines of the sea cucumber parent, remove the gonads and cut or crush them to release the eggs or sperm, thereby activating the eggs and sperm respectively;

[0012] D. Fertilization and Incubation: Mix the eggs and sperm and wash the eggs. Collect the fertilized eggs for incubation. Control the incubation density and continuously aerate to keep the fertilized eggs suspended. When the fertilized eggs develop into small, ear-shaped larvae that can open their mouths to feed, separate them into ponds for processing.

[0013] E. Planktonic Larvae Cultivation: Planktonic larvae are cultivated in a closed indoor environment, with controlled density and aeration to keep the larvae suspended. The water is changed daily, and Chaetoceros algae and baker's yeast are fed. Dead larvae, leftover bait, and feces are regularly removed from the pond bottom.

[0014] F. Larval attachment and cultivation: When more than 30% of the planktonic larvae develop into the barrel-shaped larvae stage, the attachment substrate is released. After release, the water is changed every day and Chaetoceros algae, baker's yeast, and juvenile sea cucumber feed are fed. The amount of juvenile sea cucumber feed increases as the fry grows, while the amount of Chaetoceros algae and baker's yeast gradually decreases. When all the planktonic larvae are attached, the Chaetoceros algae and baker's yeast feeding is stopped, and cultivation is continued to obtain large-sized tropical sea cucumber fry;

[0015] G. Intermediate growth of tropical sea cucumbers: When tropical sea cucumber seedlings grow to 10-20mm, they are collected and transferred to outdoor cages for cultivation. The cages are built in advance and naturally attached benthic diatoms are used as bait. During the intermediate growth period, additional artificial compound bait can be fed to increase the growth rate of the young sea cucumbers, and large-sized sea cucumbers can be continuously cultivated.

[0016] Preferably, the chopping in step C is to cut the female gonad into small segments less than 5 mm.

[0017] Preferably, the crushing in step C is crushing the male gonads.

[0018] Preferably, the gonads are removed in step C by temporarily keeping the parent ginseng to empty its intestines, then making a small incision from the tail and removing the gonads from the opening, while trying not to damage the body wall and other organs during the process, thereby reducing damage to the parent ginseng.

[0019] Preferably, the egg and sperm activation in step C is performed by soaking the egg in the seawater of step A for 30-60 minutes and soaking the sperm in the seawater of step A for 5-15 minutes. The standard for successful egg activation is that the egg becomes round in shape and the germinal vesicle ruptures.

[0020] Preferably, the tropical sea cucumber is sea cucumber of Yuzu foot, sea cucumber of brown ring or sea cucumber of Mie.

[0021] Preferably, the Chaetoceros and baker's yeast are Chaetoceros and baker's yeast used to prepare bait, wherein the density of Chaetoceros is not less than 2 million / mL, the baker's yeast is crushed into a 0.1g / L suspension using a 200-mesh sieve in clean seawater, and then the Chaetoceros liquid and the baker's yeast suspension are mixed in a volume ratio of 3:1; the Chaetoceros is Chaetoceros muelleri in the exponential growth period; and the juvenile sea cucumber bait is an imitation sea cucumber juvenile sea cucumber bait.

[0022] Preferably, the Chaetoceros muelleri is prepared by the following method: the Chaetoceros muelleri is cultured separately using F / 2 culture medium, and a three-stage culture is adopted, wherein the first-stage seed is preserved in a 5L sterilized conical flask in a constant temperature workshop, the second-stage seed expansion is cultured in a 15L transparent plastic barrel, and the third-stage culture is completed using a 200-400L plastic barrel; wherein the seawater used for the first and second-stage cultures of the F / 2 culture medium is boiled and then cooled to room temperature before use; the third-stage culture is carried out in a 200-400L plastic barrel, 20ppm disinfectant powder is added to the seawater overnight, and then neutralized with 10ppm sodium thiosulfate before use, and the Chaetoceros muelleri culture is carried out indoors, using a 20-50 watt full-spectrum fluorescent lamp to continuously provide 10,000-30,000lx of light, and at night, the plastic barrel used for the third-stage culture is covered with a transparent acrylic plate for full protection.

[0023] Preferably, the method for artificially breeding tropical sea cucumbers is as follows:

[0024] A. Water Quality Control: Natural seawater is filtered and / or disinfected with UV light before use. The first stage of the two-stage filtration is a standard sand filter to remove large particles from the natural seawater. The second stage uses filter bags with a pore size of no more than 5 microns to further remove fine particles. To remove pathogens from the seawater, the seawater is disinfected with UV light before and after each stage of filtration. The salinity of the treated seawater is between 29-33ppt and the temperature is between 26-31°C, resulting in qualified treated seawater.

[0025] B. Collection of sea cucumber parents: In the season when tropical sea cucumber gonads mature, wild tropical sea cucumbers are collected in natural sea areas as broodstock;

[0026] C. Gamete acquisition: After temporarily keeping the parent ginseng to empty its intestines, a small incision is made from the tail and the gonads are removed from the opening. Try not to damage the body wall and other organs during the process to reduce damage to the parent ginseng;

[0027] Egg collection method: Select female ginseng with well-developed gonads, dissect and obtain the gonads. Cut the gonads into small segments <5mm, allow the eggs to flow out naturally, and then dry in the shade for 1-2 hours. Filter through a 200-mesh screen to remove large tissue fragments. Concentrate the filtrate through a 300-mesh screen to remove small tissue fragments. Then, soak the eggs in seawater for 30-60 minutes to activate the eggs. The standard for successful egg activation is rounding and rupture of the germinal vesicle.

[0028] Sperm collection method: Select male ginseng with well-developed gonads, dissect and obtain the gonads, crush them, and filter through a 400-mesh screen to remove large tissue fragments. Place the filtrate in clean seawater and let it stand for 5-15 minutes to activate the sperm. Examine under a microscope and select the sperm with high activity for use.

[0029] D. Fertilization and Incubation: Collect eggs in a 5L plastic measuring cup, add fresh seawater, and then add sperm. Stir, let stand, and remove the supernatant. Repeat three times to remove excess sperm, impurities such as poorly fertilized eggs, and other impurities. Add the collected fertilized eggs to the incubation tank at a density of 3-5 eggs / mL. Continuously aerate the tank during incubation to ensure uniform suspension of the fertilized eggs. Incubate the fertilized eggs at a temperature of 28-31°C and a salinity of 29-33 ppt, changing the water volume by 1 / 4-1 / 2 daily. Incubate for two days until the fertilized eggs develop into small, ear-shaped larvae capable of feeding. Then, separate the eggs into separate tanks. The larvae density after separation should not exceed 1 egg / mL.

[0030] E. Planktonic larvae cultivation: Planktonic larvae are cultivated in a closed indoor environment, with controlled density and proper aeration throughout the cultivation process to keep the larvae in a suspended state.

[0031] The cultivation of sea cucumber planktonic larvae is divided into two stages, with the middle ear larvae as the boundary. The first stage is from the development of small ear larvae to the middle ear larvae. 1 / 3 of the water is changed every day. Chaetoceros algae and baker's yeast are fed once in the morning and afternoon after the water is changed. The feeding amount is 5L / m 3 Water body, the daily feeding amount is 10L / m 3 Water body; The second stage is after the middle ear larvae, including the large ear larvae to the bottle larvae stage. Change 1 / 2 of the water every day. Feed Chaetoceros algae and baker's yeast once in the morning and afternoon after changing the water. The feeding amount each time is 10L / m 3 Water body, the daily feeding amount is 20L / m 3 Water: Use silk screen to filter when changing water to prevent the escape of sea cucumber planktonic larvae. Use siphoning to suck the bottom of the pond every three days to remove dead larvae and residual bait and feces.

[0032] F. Larval attachment and cultivation: When more than 30% of the planktonic larvae have developed to the bottle-shaped larvae stage, begin adding attachment substrates. These are polyethylene corrugated sheets for attaching benthic diatoms. 10-15 sets of attachment substrates are added per cubic meter of water. After addition, change half the water daily. Continue feeding Chaetoceros and baker's yeast after water changes in the early morning and afternoon. The amount of feed decreases as the density of planktonic larvae in the water decreases. Additionally, feed the juvenile sea cucumber feed, containing 5 ppm of nutrient, once each morning and evening. Before use, crush the juvenile sea cucumber feed using a 200-mesh silk sieve in clean seawater to form a suspension.

[0033] When all the planktonic larvae have attached, stop feeding Chaetoceros and baker's yeast, and instead feed the young sea cucumbers with mixed feed. The amount of feed increases as the seedlings grow. When the seedlings reach a length of more than 2 mm, remove the remaining bait and feces from the bottom of the pond once a week by siphoning. Change the water once a day in the morning and evening, changing 1 / 2 of the water each time. Feed the fish after each water change, and increase the feeding amount to 20ppm each time. Continue to cultivate and obtain large-sized tropical sea cucumber seedlings.

[0034] G. Intermediate growth of tropical sea cucumbers: When tropical sea cucumber seedlings grow to 10-20mm, they are transferred to sea cages for intermediate growth. The cages are nylon mesh cages with a length of 1m, a width of 1m and a height of 1m, and a mesh size of 2mm. The cages need to be arranged one week in advance to attach benthic diatoms as bait; check the cages weekly and remove pests. If the culture density is too high, additional feed can be added until large-sized sea cucumbers are cultivated for bottom seeding.

[0035] Preferably, when harmful organisms break out in the water body where sea cucumber larvae are cultured, 2-3 ppm of trichlorfon is added to kill them, and the water is completely changed 2 hours after the addition of trichlorfon; in order to inhibit the growth of pathogens, 2-3 ppm of antibacterial drugs are added every 3 days during the seedling period.

[0036] The present invention also provides application of the above-mentioned tropical sea cucumber artificial breeding method in breeding tropical sea cucumbers.

[0037] Preferably, the tropical sea cucumber is sea cucumber of Yuzu foot, sea cucumber of brown ring or sea cucumber of Mie.

[0038] The water quality control method of the present invention requires that the natural seawater used for seedling cultivation must undergo two-stage filtration and / or ultraviolet disinfection before use. The pest and disease control method of the present invention firstly removes insect eggs and pathogens through filtration and / or ultraviolet disinfection. The addition of microporous filter bags to the seedling cultivation water further removes insect eggs and other microscopic particles from the seawater, thus eliminating the outbreak of pests at the source. Secondly, the introduction of pests through bait is prevented by controlling the cultivation of unicellular algae. Thirdly, drugs are added to the seedling cultivation system to kill pests and control the growth of pathogens.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) Compared with existing traditional tropical sea cucumber artificial induction methods such as shade drying, running water stimulation, heat stimulation, etc., the dissection method requires very few parental parasites and can obtain gametes and fertilized eggs every time, while other methods require a large number of parental parasites and may not be able to successfully induce labor every time. Therefore, the success rate of artificial breeding using the dissection method is higher. The present invention has strong operability, is easy to promote, and can be used for large-scale production of tropical sea cucumber seedlings, and is particularly suitable for species that are not easy to induce labor. This method provides a basis for tropical sea cucumber resource recovery, conventional breeding, and polyploid breeding.

[0041] (2) The gonad extraction method used in the present invention does not damage the body wall, organs, and other key structures of the parent, and the parent maintains growth vitality after gonad extraction. The gonad extraction process of the present invention causes a small incision, and the sea cucumber is less damaged, so it recovers quickly and does not affect the continued artificial breeding of the parent ginseng in the second year.

[0042] (3) The cultivation of the unicellular algae bait of the present invention is carried out entirely under indoor conditions, using a full-spectrum plant growth lamp as the light source, and covered with a colorless, transparent acrylic sheet to prevent flying insects and other pollutants from entering the cultivation system. Compared with traditional bait algae cultivation methods, this method is not affected by natural light, and the unicellular algae can grow continuously for 24 hours, with a growth rate at least twice that of traditional unicellular algae cultivation methods. It is also immune to the influence of weather, temperature, pollutants, and flying insects, and can provide a stable and efficient supply of fresh bait for sea cucumber larvae.

[0043] (4) The present invention breaks the concept that tropical sea cucumbers cannot be artificially bred through dissection. For the first time, several tropical sea cucumber species such as the jade-foot sea cucumber, the brown-ringed sea cucumber, and the Mi's sea cucumber were bred through dissection. This method can provide a technical guarantee for the continuation and resource recovery of tropical sea cucumber populations that are on the verge of extinction and cannot be artificially induced to produce.

[0044] (5) The artificial breeding of tropical sea cucumbers using this method is simple and easy to operate. Through processes such as water quality control, parent collection, gamete acquisition, fertilization and hatching, and larval cultivation, it can effectively overcome a series of problems in the seedling cultivation process, such as the inability to induce labor / poor induction of labor, unstable output, and unscientific feeding of bait, thereby cultivating a large number of tropical sea cucumber offspring and improving the success rate of artificial breeding of tropical sea cucumber seedlings.

[0045] (6) The present invention has the advantages of being easy to operate, highly practical, and easy to promote on a large scale. Moreover, the method will not damage the key structures of the parent organism, which provides the possibility for conventional quantitative genetic breeding and chromosome manipulation breeding of tropical sea cucumbers. It also lays a theoretical and practical foundation for the recovery of such sea cucumber resources, and has a very broad application space in the field of artificial breeding and aquaculture of tropical sea cucumbers. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Microporous filter bags for filtering seawater.

[0047] Figure 2 Figures 1 and 2 show the primary, secondary, and tertiary cultures of Chaetoceros muelleri. The primary culture was in a 5-L sterile conical flask (A), the secondary culture was in a 15-L transparent plastic bucket (B), and the tertiary culture system was covered with a plexiglass lid to prevent contamination by flying insects (C).

[0048] Figure 3 The female gonads (A) and male gonads (B) of the dissected sea cucumber.

[0049] Figure 4 The female gonads of the sea cucumber are cut into small pieces.

[0050] Figure 5Immature sea cucumber eggs (A) and mature sea cucumber eggs (B).

[0051] Figure 6 The small-ear larvae (A), late middle-ear larvae (B), large-ear larvae (C), and bottle-shaped larvae (D) of the sea cucumber obtained by dissection.

[0052] Figure 7 The body of the sea cucumber seedling grows to 10-20mm.

[0053] Figure 8 The female gonads (A) and male gonads (B) of the brown-ringed sea cucumber obtained by dissection.

[0054] Figure 9 The female gonads (A) and male gonads (B) of the sea cucumber Misaki obtained by dissection.

[0055] Figure 10 The female gonads of the brown-ringed sea cucumber are cut into small pieces.

[0056] Figure 11 The female gonads of the sea cucumber Misaki are cut into small pieces.

[0057] Figure 12 The small-ear larvae (A), late middle-ear larvae (B), large-ear larvae (C), and bottle-shaped larvae (D) of the brown-ringed sea cucumber obtained by dissection.

[0058] Figure 13 The small-ear larvae (A), late middle-ear larvae (B), large-ear larvae (C), and barrel-shaped larvae (D) of the sea cucumber Mieke obtained by dissection.

[0059] Figure 14 The body length of the brown-ringed sea cucumber seedlings reaches 10-20mm.

[0060] Figure 15 The body length of the Misaki sea cucumber seedlings reaches 10-20mm. DETAILED DESCRIPTION

[0061] The present invention is further described below with reference to specific examples, which do not limit the present invention in any way. Unless otherwise specified, the methods and devices used in the present invention are commonly used in the art.

[0062] Example 1: Artificial breeding of sea cucumbers

[0063] Starting from July 30, 2022, the artificial breeding experiment of sea cucumbers was carried out at the Daya Bay Marine Biological Comprehensive Experiment Station of the Chinese Academy of Sciences in Shenzhen using the method of the present invention. The relevant process is as follows:

[0064] (1) Water quality control:

[0065] The natural seawater used for seedling cultivation is taken from the sea area near the experimental station. After monitoring, the content of various pollutants is lower than the second-class seawater quality standard. The extracted seawater is filtered through sand and then filtered through a 5-micron filter bag ( Figure 1 Because the local seawater contained relatively few pathogens and other contaminants, UV sterilization was not performed. During this experiment, the treated seawater had a salinity range of 29-33 ppt and a temperature range of 28-31°C, resulting in seawater suitable for larval aquaculture.

[0066] (2) Unicellular algae bait culture:

[0067] The F / 2 culture medium was used to culture Chaetoceros muelleri, and a three-stage culture was used. Among them: the first-stage culture was kept in a 5L sterilized conical bottle in a constant temperature workshop ( Figure 2 A), the secondary expansion is cultured in 15L transparent plastic barrels ( Figure 2 B), the third level culture is carried out using 200-400L white plastic barrels ( Figure 2 C). The seawater used in the primary and secondary F / 2 culture medium is boiled and then cooled to room temperature. For the tertiary culture, seawater is incubated overnight with 20 ppm disinfectant powder and then neutralized with 10 ppm sodium thiosulfate. To avoid the hazards of outdoor cultivation, such as contamination, wind and rain, unstable light, and high temperatures, Chaetoceros muelleri cultivation is performed indoors. Full-spectrum fluorescent lamps (20-50 watts, 10,000-30,000 lux) provide continuous illumination. At night, the plastic buckets used for tertiary cultivation are covered with acrylic sheets for full protection. This method of cultivation accelerates the growth of the microalgae. Depending on the inoculum density, Chaetoceros muelleri typically reaches exponential growth within 2-3 days. This cultivation method is independent of natural light and allows for rapid, continuous growth around the clock. It protects against the hazards of outdoor cultivation, such as wind, rain, and high temperatures, as well as predators such as protozoa. It provides a stable supply of fresh, live algae for sea cucumber larvae, and feeding them directly does not introduce diseases or predators.

[0068] (3) Fertilized egg acquisition:

[0069] In the season when tropical sea cucumber gonads mature, wild tropical sea cucumbers are collected from natural sea areas as broodstock. The broodstock of Yuzu sea cucumber is collected from Daya Bay waters, temporarily kept for 3 days to adapt to the indoor environment and empty the intestines, and then the female gonads of Yuzu sea cucumber are obtained by the method of the present invention ( Figure 3 A) and male gonads ( Figure 3 B): Make a small incision at the tail and remove the gonads from the opening, trying not to damage the body wall or other organs to minimize damage to the ginseng.

[0070] Cut the female gonad into small pieces <5 mm ( Figure 4), let the eggs flow out naturally, most of the eggs that flow out have not yet been activated, and the germinal vesicles are still there ( Figure 5 A). Dry in the shade for 2 hours to break the germinal vesicles, filter through a 200-mesh screen to remove large tissue fragments, and concentrate the filtrate through a 300-mesh screen to remove small tissue fragments. Then, soak in clean seawater for 30-60 minutes to activate the eggs. The standard for successful egg activation is that the egg shape becomes round and the germinal vesicles break ( Figure 5 B).

[0071] Select male ginseng with well-developed gonads, dissect and obtain the gonads, crush them and filter them through a 400-mesh screen to remove large tissue pieces. Place the filtrate in clean seawater and let it stand for 5-15 minutes to activate the sperm. Examine under a microscope and select the sperm with high activity for use.

[0072] After the eggs are concentrated, they are collected in a 5L plastic measuring cup, fresh seawater is added, and sperm is added; stirring, standing, and the supernatant is removed. This process is repeated 3 times to remove impurities such as excess sperm and poorly fertilized eggs; then the collected fertilized eggs are added to several 1ml 3 In the hatching pond, the density of fertilized eggs is 3-5 eggs / mL. During the incubation period, aeration is continuously carried out to prevent the fertilized eggs from sinking to the bottom. The fertilized eggs are kept at a temperature of 28-31°C and a salinity of 29-33ppt. About 1 / 3 of the water is changed every day. After 2 days of incubation, when the fertilized eggs develop into small ear-shaped larvae that can open their mouths to eat, they are separated into ponds. The density of the larvae after separation is 1 egg / mL.

[0073] (4) Planktonic larvae cultivation:

[0074] The planktonic larvae of the sea cucumber are cultivated in a closed indoor environment with a light intensity not exceeding 1000lx, a temperature of 28-31℃, and a salinity of 29-33ppt. Moderate aeration is maintained throughout the cultivation process to keep the larvae in a suspended state.

[0075] The cultivation of planktonic larvae of sea cucumbers is divided into two stages, with the middle ear larvae as the boundary. The first stage is the small ear larvae opening their mouths to feed ( Figure 6 A) to the late middle ear larvae ( Figure 6 B) Change 1 / 3 of the water every day, feed the mixture of Chaetoceros muelleri and baker's yeast once in the morning and afternoon after changing the water, and feed 5L / m 3 Water body, the daily feeding amount is 10L / m 3 Water; the second stage is after the middle ear larvae, including the large ear larvae ( Figure 6 C) to bottle-shaped larvae ( Figure 6 In the D) stage, 1 / 2 of the water was changed every day, and a mixture of Chaetoceros muelleri and baker's yeast was fed once in the morning and afternoon after the water was changed. The feeding amount each time was 10L / m 3 Water body, the daily feeding amount is 20L / m 3When changing the water, use a sieve of appropriate size based on the development of the larvae to filter the water to prevent planktonic larvae from escaping. Siphon the bottom of the pond every three days to remove dead larvae, residual bait, and feces. During the cultivation of planktonic larvae, measure survival rate, body length, and water quality every three days. If pests such as copepods and mosquito larvae occur in the water for sea cucumber larvae cultivation, add 2-3 ppm of trichlorfon to kill them. Completely change the water two hours after adding trichlorfon. To inhibit the growth of pathogens, add 2-3 ppm of penicillin or other antimicrobial drugs every three days during the nursery period.

[0076] Mixture of Chaetoceros muelleri and baker's yeast: Prepare bait by combining Chaetoceros muelleri in the exponential growth period with baker's yeast, wherein the density of Chaetoceros muelleri is not less than 2 million / mL. Use a 200-mesh sieve to rub the baker's yeast into a 0.1g / L suspension in clean seawater. Then mix the Chaetoceros muelleri liquid and the baker's yeast suspension in a volume ratio of 3:1 and feed it as bait.

[0077] (5) Larvae attachment and cultivation:

[0078] When more than 30% of the planktonic sea cucumber larvae have reached the barrel-shaped stage, cultchnose substrates are introduced. These consist of corrugated polyethylene sheets for benthic diatoms. Ten to 15 sets (31 cm x 39 cm, 20 sheets / set) are placed per cubic meter of water. Half the water is changed daily after introduction. A mixture of Chaetoceros muelleri and baker's yeast is continued after water changes in the morning and afternoon. The feed rate decreases as the density of planktonic larvae decreases. During water changes, the fish are filtered using a sieve of appropriate size, depending on the development of the larvae. Siphoning the bottom of the pond is performed every three days to remove dead larvae, residual feed, and feces. In addition, a 5 ppm commercially available sea cucumber juvenile feed is fed once each morning and evening. This feed is crushed with a 200-mesh sieve in treated seawater before use to create a suspension. When all the larvae are attached (based on the absence of planktonic larvae in the nursery water), stop adding the mixture of Chaetoceros muelleri and baker's yeast, and instead feed the sea cucumber juveniles with bait. The amount of feed increases as the seedlings grow, and the tropical sea cucumber seedlings are continuously cultivated. When the seedlings are more than 2 mm in length, remove the residual bait and feces from the bottom of the pond once a week by siphoning. Change the water once a day in the morning and evening, changing 1 / 2 of the water each time, and feed the fish after the water change. The amount of feed is increased to 20 ppm each time, depending on the amount of seedlings attached. When harmful organisms break out in the water body where the sea cucumber larvae are cultured, the treatment method is as in step (4).

[0079] (6) Intermediate cultivation of tropical sea cucumbers:

[0080] When the ginseng seedlings reach 10-20mm in length ( Figure 7) can be transferred to outdoor sea cages for intermediate grow-out. These cages are nylon mesh cages, 1m long, 1m wide, and 1m high, with a 2mm mesh size. The cages need to be set up a week in advance and fed with naturally attached benthic diatoms. The cages need to be inspected weekly, and any predators found should be removed immediately. During the intermediate grow-out period, additional artificial feeds can be fed to increase growth and continuously cultivate larger sea cucumbers.

[0081] The development speed of juvenile sea cucumbers bred by the dissection method and the shade drying method (CN102090363A) was compared (Table 1). During the experiment, it was found that there was no significant difference in the development speed of juvenile sea cucumbers bred by the two methods. The fertilized eggs of the juvenile sea cucumbers bred by the dissection method successfully hatched into small-eared juveniles after about 36 hours ( Figure 6 A), about 5 days after fertilization, the larva gradually grows larger, grows new body arms, and develops into a middle ear-shaped larva ( Figure 6 B), 10-14 days after fertilization, the larva develops into the large-eared larva stage ( Figure 6 C), the planktonic stage larvae reach their maximum size and grow a spherical body that plays a very important role in the metamorphosis of sea cucumber larvae. About 14-20 days after fertilization, the body and body of the late stage larvae of the big ear larvae begin to undergo significant changes. The body size shrinks sharply and the larvae develop into a barrel-shaped larvae ( Figure 6 D), gradually lose the ability to float, and the larvae also change from the floating state to the attached state. 90 days after fertilization, when the ginseng seedlings reach 10-20mm ( Figure 7 ) and above, and then transferred to outdoor cages for intermediate growth.

[0082] Table 1 Development timeline of sea cucumber from fertilized egg to juvenile

[0083]

[0084]

[0085] Example 2: Artificial breeding of brown-ringed sea cucumbers and Mi's sea cucumbers

[0086] Starting from July 30, 2022, artificial breeding experiments of brown-ringed sea cucumbers and Mieke's sea cucumbers were carried out at the Daya Bay Marine Biological Comprehensive Experimental Station of the Chinese Academy of Sciences in Shenzhen using the method of the present invention. At present, artificial induction and breeding of these two sea cucumbers have not been successfully carried out. The relevant process is as follows (the breeding method of the two sea cucumbers is the same, except that the different types of sea cucumbers are handled separately. The water quality control and unicellular algae bait culture during the breeding process are as in Example 1):

[0087] (1) Fertilized egg acquisition:

[0088] Two tropical sea cucumbers were collected from Daya Bay. After being temporarily kept for 3 days to adapt to the indoor environment and empty their intestines, the female gonads of the brown ring sea cucumber were obtained by dissection ( Figure 8 A) and male gonads ( Figure 8 B), Female gonads of Misaki sea cucumber ( Figure 9 A) and male gonads ( Figure 9 B): Make a small incision at the tail and remove the gonads from the opening, trying not to damage the body wall or other organs to minimize damage to the ginseng.

[0089] Cut the female gonad into small pieces <5 mm ( Figure 10 、 Figure 11 ) and allow the eggs to flow naturally. Most of the released eggs are unactivated, and the germinal vesicles remain. Dry in the shade for 2 hours to rupture the germinal vesicles. Filter through a 200-mesh silk screen to remove large tissue fragments. Concentrate the filtrate through a 300-mesh silk screen to remove small fragments. Then, soak in clean seawater for 30-60 minutes to activate the eggs. Successful egg activation is indicated by a rounded appearance and rupture of the germinal vesicles.

[0090] Select male ginseng with well-developed gonads, dissect and obtain the gonads, crush them and filter them through a 400-mesh screen to remove large tissue pieces. Place the filtrate in clean seawater and let it stand for 5-15 minutes to activate the sperm. Examine under a microscope and select highly active sperm for use.

[0091] After the eggs are concentrated, they are collected in a 5L plastic measuring cup, fresh seawater is added, and sperm is added; stirring, standing, and the supernatant is removed. This process is repeated 3 times to remove impurities such as excess sperm and poorly fertilized eggs; then the collected fertilized eggs are added to several 1ml 3 In the hatching pond, the density of fertilized eggs is 3-5 eggs / mL. During the incubation period, aeration is continuously carried out to prevent the fertilized eggs from sinking to the bottom. The fertilized eggs are kept at a temperature of 28-31°C and a salinity of 29-33ppt. About 1 / 3 of the water is changed every day. After 2 days of incubation, when the fertilized eggs develop into small ear-shaped larvae that can open their mouths to feed, they are separated into ponds. The density of the larvae after separation is 1 egg / mL.

[0092] (2) Planktonic larvae cultivation:

[0093] Sea cucumber planktonic larvae are cultivated in a closed indoor environment with a light intensity not exceeding 1000lx, a temperature of 28-31℃, and a salinity of 29-33ppt. Moderate aeration is maintained throughout the cultivation process to keep the larvae in a suspended state.

[0094] The cultivation of sea cucumber planktonic larvae is divided into two stages, with the middle ear larvae as the boundary. The first stage is before the development of the small ear larvae to the middle ear larvae. 1 / 3 of the water is changed every day. In the morning and afternoon, after the water is changed, the mixture of Chaetoceros muelleri and baker's yeast (as described in Example 1) is fed once, and the feeding amount is 5L / m3 Water body, the daily feeding amount is 10L / m 3 The second stage is from the middle ear larvae to the bottle-shaped larvae. Change 1 / 2 of the water every day. Feed the fish with a mixture of Chaetoceros muelleri and baker's yeast once in the morning and afternoon after changing the water. The feeding amount is 10L / m 3 Water body, the daily feeding amount is 20L / m 3 When changing the water, use a sieve of appropriate size based on the development of the larvae to filter the water to prevent the escape of planktonic larvae. Siphon the bottom of the pond every three days to remove dead larvae, residual bait, and feces. During the cultivation of planktonic larvae, measure the survival rate, body length, and water quality every three days. If pests such as copepods and mosquito larvae occur in the water for sea cucumber larvae cultivation, add 2-3 ppm of trichlorfon to kill them. Completely change the water two hours after adding trichlorfon. To inhibit the growth of pathogens, add 2-3 ppm of penicillin or other antimicrobial drugs every three days during the larval stage.

[0095] (3) Larvae attachment and cultivation:

[0096] When more than 30% of the planktonic sea cucumber larvae have reached the barrel-shaped stage, cultchnose substrates are introduced. These consist of corrugated polyethylene sheets for benthic diatoms. Ten to 15 sets (31 cm x 39 cm, 20 sheets / set) are placed per cubic meter of water. After introduction, half of the water is changed daily. A mixture of Chaetoceros muelleri and baker's yeast is continued after water changes in the morning and afternoon. The feed rate decreases as the density of planktonic larvae decreases. During water changes, the fish are filtered using a sieve of appropriate size, depending on the development of the larvae. Siphoning the bottom of the pond is performed every three days to remove dead larvae, residual feed, and feces. In addition, a 5 ppm commercially available sea cucumber juvenile feed is fed once each morning and evening. The juvenile feed is crushed with a 200-mesh sieve in treated seawater before use to create a suspension. When all the larvae are attached (based on the absence of planktonic larvae in the nursery water body), stop adding the mixture of Chaetoceros muelleri and baker's yeast, and instead feed the young sea cucumbers with bait. The amount of feed increases as the seedlings grow, and the tropical sea cucumber seedlings are continuously cultivated. When the seedlings reach a length of more than 2 mm, remove the residual bait and feces from the bottom of the pond once a week by siphoning. Change the water once a day in the morning and evening, changing 1 / 2 of the water each time, and feed the fish after the water change. The amount of feed is increased to 20 ppm each time, depending on the amount of seedlings attached. When the water body for the cultivation of sea cucumber larvae breaks out with harmful organisms, the treatment method is as in step (2).

[0097] (4) Intermediate cultivation of tropical sea cucumbers:

[0098] When the sea cucumber seedlings reach a length of 10-20 mm or more, they can be transferred to outdoor sea cages for intermediate ongrowing. These cages are nylon mesh cages, 1 meter long, 1 meter wide, and 1 meter high, with a 2mm mesh size. The cages should be set up a week in advance to allow for the accumulation of a sufficient number of benthic diatoms. The cages should be inspected weekly, and any predators detected should be removed immediately. If the stocking density is too high, additional artificial feed may be added until the sea cucumbers have reached a large size suitable for bottom seeding.

[0099] Comparing the developmental speed of brown-ringed sea cucumbers bred by dissection and those of Mieke’s sea cucumber larvae (Table 2), it was found that the developmental speed of the two sea cucumber larvae was almost the same. The fertilized eggs of brown-ringed sea cucumbers bred by dissection successfully hatched into small-eared larvae ( Figure 12 A) The Misaki sea cucumber hatches successfully into small-eared larvae in about 42 hours ( Figure 13 A). About 5 days after fertilization, the larvae gradually grow larger and grow new arms. Both develop into middle ear-shaped larvae ( Figure 12 B, Figure 13 B). 10-14 days after fertilization, the larva develops into a large-eared larva ( Figure 12 C, Figure 13 C), the planktonic stage larvae reach their maximum size and grow a spherical body that plays a very important role in the metamorphosis of sea cucumber larvae. About 15 days after fertilization, the body and body of the late stage larvae of the big ear larvae begin to undergo significant changes. The body size shrinks sharply and the larvae develop into a barrel-shaped larvae ( Figure 12 D, Figure 13 D), gradually lose the ability to float, and the larvae also change from the floating state to the attached state. 90 days after fertilization, when the ginseng seedlings reach 10-20mm ( Figure 14 , Figure 15 ) and above, and then transferred to outdoor cages for intermediate growth.

[0100] Table 2 Development timeline of brown-ringed sea cucumber and Mieke's sea cucumber from fertilized eggs to juveniles

[0101]

[0102]

[0103] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for artificial breeding of tropical sea cucumbers, characterized in that: The following steps are involved: A. Water Quality Control: Natural seawater is filtered and disinfected with UV light before use. The first stage of the two-stage filtration involves ordinary sand filtration to remove large particles from the natural seawater. The second stage involves filtration using filter bags with a pore size of no more than 5 microns to further remove fine particles. To remove pathogens from the seawater, the seawater is disinfected with UV light before and after each stage of filtration. The salinity of the treated seawater is between 29-33 ppt and the temperature is between 26-31°C, resulting in qualified treated seawater. B. Collection of sea cucumber parents: In the season when tropical sea cucumber gonads mature, wild tropical sea cucumbers are collected in natural sea areas as broodstock; C. Gamete acquisition: After temporarily keeping the parent ginseng to empty its intestines, a small incision is made from the tail and the gonads are removed from the opening. Try not to damage the body wall and other organs during the process to reduce damage to the parent ginseng; Egg collection method: Select female ginseng with well-developed gonads, dissect and obtain gonads, cut the gonads into small segments <5 mm, allow the eggs to flow out naturally, and then dry in the shade for 1-2 hours. Filter through a 200-mesh screen to remove large tissue fragments. Concentrate the filtrate through a 300-mesh screen to remove small tissue fragments. Then, soak in the qualified seawater for 30-60 minutes to activate the eggs. The standard for successful egg activation is rounding and rupture of the germinal vesicle. Sperm collection method: Select male ginseng with well-developed gonads, dissect and obtain the gonads, crush them, filter them through a 400-mesh screen to remove large tissue fragments, place the filtrate in the above-mentioned qualified seawater and let it stand for 5-15 minutes to activate the sperm, examine it under a microscope and select the sperm with high activity for use; D. Fertilization and incubation: Collect the eggs in a 5 L plastic measuring cup, add fresh seawater, and add sperm; Stir, let stand, and remove the supernatant. Repeat this process three times to remove excess sperm and impurities. The collected fertilized eggs are then added to the incubation tank at a density of 3-5 eggs / mL. Aeration is continuously maintained during incubation to ensure uniform suspension of the eggs. The fertilized eggs are incubated at a temperature of 28-31°C and a salinity of 29-33 ppt, with a daily water change of 1 / 4-1 / 2. After two days of incubation, when the fertilized eggs develop into small, ear-shaped larvae capable of feeding, they are separated into separate tanks. The larvae density after separation should not exceed 1 egg / mL. E. Planktonic larvae cultivation: Planktonic larvae are cultivated in a closed indoor environment, with controlled density and proper aeration throughout the cultivation process to keep the larvae in a suspended state. The cultivation of sea cucumber planktonic larvae is divided into two stages, with the late middle ear larvae as the boundary. The first stage is from the development of small ear larvae to the late middle ear larvae. One-third of the water is changed daily, and Chaetoceros algae and baker's yeast are fed once in the morning and afternoon after the water change. The feeding amount is 5 L / m³ of water each time, and the total daily feeding amount is 10 L / m³ of water. The second stage is from the late middle ear larvae to the bottle larvae. One-half of the water is changed daily, and Chaetoceros algae and baker's yeast are fed once in the morning and afternoon after the water change. The total daily feeding amount is 20 L / m³ of water. When changing the water, a silk screen is used to filter to prevent the planktonic sea cucumber larvae from escaping. The bottom of the pond is siphoned every three days to remove dead larvae, residual bait, and feces. F. Larval attachment and cultivation: When more than 30% of the planktonic larvae have developed into the bottle-shaped larvae stage, begin adding attachment substrates. These are polyethylene corrugated sheets for attaching benthic diatoms. 10-15 sets of attachment substrates are added per cubic meter of water. After addition, change half of the water daily. Continue feeding Chaetoceros and baker's yeast after water changes in the early morning and afternoon. The amount of feed decreases as the density of planktonic larvae decreases. Additionally, feed the fish once in the morning and evening a 5 ppm juvenile sea cucumber feed. Before use, crush the juvenile sea cucumber feed in clean seawater using a 200-mesh silk sieve to form a suspension. When all the planktonic larvae have attached, stop feeding Chaetoceros and baker's yeast, and instead feed the juvenile sea cucumber feed. The amount of feed increases as the fry grows. When the fry reach a length of more than 2 mm, remove the residual bait and feces from the bottom of the pond once a week by siphoning. Change the water once a day in the morning and evening, changing 1 / 2 of the water each time. Feed the fish after each water change, and increase the feeding amount to 20ppm each time. Continue to cultivate large-sized tropical sea cucumber fry. Intermediate growout of tropical sea cucumbers: When tropical sea cucumber seedlings grow to 10-20 mm, they are transferred to sea cages for intermediate growout. The cages are nylon mesh cages 1 m long, 1 m wide, and 1 m high, with a mesh size of 2 mm. The cages need to be set up a week in advance and are fed with benthic diatoms. The cages are inspected weekly and pests removed. If the stocking density is too high, additional feeding is applied until the sea cucumbers are large enough for bottom seeding. The tropical sea cucumber is sea cucumber of jade foot, sea cucumber of brown ring or sea cucumber of Mie.

2. The method for artificial breeding of tropical sea cucumbers according to claim 1, wherein The Chaetoceros and baker's yeast are prepared by combining Chaetoceros with baker's yeast to prepare bait, wherein the density of Chaetoceros is not less than 2 million / mL, the baker's yeast is crushed into a 0.1 g / L suspension using a 200-mesh sieve in clean seawater, and then the Chaetoceros liquid and the baker's yeast suspension are mixed in a volume ratio of 3:1; the Chaetoceros is Chaetoceros muelleri in the exponential growth period; and the juvenile sea cucumber bait is an imitation sea cucumber juvenile sea cucumber bait.

3. Application of the method for artificial breeding of tropical sea cucumbers according to claim 1 in breeding tropical sea cucumbers; the tropical sea cucumbers are sea cucumbers of the jade foot, sea cucumbers of the brown ring or sea cucumbers of Mie.

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