High-efficiency artificial breeding and intermediate culture method of holothuria leucospilota

Abstract

This invention discloses a highly efficient artificial breeding and intermediate rearing method for *Sinocyclocheilus rubra*. Gonadally mature *Sinocyclocheilus rubra* are first air-dried, then transferred to seawater, where sodium sulfite is added to induce oxygen deprivation and stimulate spawning. Based on the physiological characteristics of *Sinocyclocheilus rubra*, this invention uses sodium sulfite to induce oxygen deprivation and asphyxiation, achieving excellent spawning-inducing results. Compared to existing traditional artificial spawning-inducing methods for tropical sea cucumbers, such as flowing water stimulation and air-drying, which have a maximum spawning rate of 13.3-18.0%, this method is significantly more effective (spawning rate as high as 75-81%). Sodium sulfite has no significant toxicity to parent sea cucumbers and is inexpensive; traditional spawning-inducing methods must be performed at night, while this invention has no time requirement, is highly operable, easy to promote, reduces the amount of parent sea cucumbers, and can provide a stable supply of fertilized eggs for large-scale sea cucumber breeding.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, and more specifically, relates to a method for the efficient artificial breeding and intermediate cultivation of sea cucumbers. Background Technology

[0002] Sea cucumbers belong to the echinoderms and are widely distributed in the intertidal zone to depths of tens of thousands of meters in the world's oceans. Except for a few planktonic and pelagic species of placoderms, the vast majority are benthic, mainly found in reefs, mud, and areas with abundant seaweed. It is estimated that there are over 1,400 species of sea cucumbers worldwide, of which more than 40 are edible. Sea cucumbers are a traditional Chinese tonic food, one of the eight treasures of the sea, and have a huge market demand in Asia. Currently, the sea cucumber with higher economic value and larger-scale farming in my country is the northern spiny sea cucumber (Apostichopus japonicus). Tropical sea cucumbers, due to a lack of artificial breeding technology and low farming efficiency, have historically relied primarily on wild-caught harvesting to meet market demand.

[0003] Sea cucumber fishing has a history of over 1,000 years. With rising human consumption levels, global sea cucumber catches have increased rapidly over the past few decades, and the world sea cucumber trade has developed into a massive industry worth billions of dollars, spanning more than 70 countries. However, under pressure from overfishing, environmental pollution, and climate change, the global sea cucumber population in natural marine areas has declined rapidly in recent years, with some species even facing extinction. In recent years, with the overexploitation of high-value sea cucumber species, some low-value species, such as the white-footed sea cucumber (Holothurialeucospilota), have been listed as important fishing targets. Due to long-term unreasonable fishing, white-footed sea cucumber resources in natural marine areas both domestically and internationally have been severely damaged. Therefore, artificial breeding and restocking are urgently needed.

[0004] Currently, one of the important ways to restore sea cucumber resources both domestically and internationally is through stock enhancement and release. Stock enhancement and release requires a stable supply of seedlings. Compared to economically important species such as the spiny sea cucumber and the rough sea cucumber (Holothuria scabra), the artificial breeding technology of the white-footed sea cucumber is still immature. Current problems include poor spawning induction effects, cumbersome operation, high costs, long planktonic larval rearing cycles, frequent predator attacks, and low metamorphosis attachment rates. Furthermore, there are no reports on the intermediate rearing of white-footed sea cucumber seedlings. To promote the industrialization of white-footed sea cucumber farming, its artificial breeding technology needs to be optimized.

[0005] Through long-term practice, we have found that the gonads of sea cucumbers in natural waters begin to mature in July and August, and their reproduction is greatly affected by time and weather. Harvesting parent sea cucumbers too early results in immature gonads, while harvesting them too late means the gonads have already matured, rendering them unsuitable for artificial breeding. Furthermore, if storms occur during the maturation of the gonads, sea cucumbers will release eggs simultaneously. Therefore, if the timing of harvesting parent sea cucumbers is not carefully controlled, a stable supply of fertilized eggs for artificial breeding cannot be provided. During the spawning induction process, we found that the success rate of spawning induction is higher on cloudy days and before storms, when air pressure decreases and dissolved oxygen concentration in the seawater drops. Therefore, a low-oxygen environment is a crucial factor in inducing spawning in sea cucumbers. Additionally, female sea cucumbers release eggs only after male sea cucumbers release sperm in the spawning induction system; therefore, sperm is also an important factor in inducing spawning. In production practice, we have also found that juvenile sea cucumbers (such as *Stichopus japonicus*) differ significantly from species like *Stichopus simus*. During the attachment and metamorphosis stage, they are more sensitive to the environment. Changes in water quality and flow caused by the introduction of corrugated attachment substrate during this stage lead to rapid sinking and mass mortality of the juveniles. Delaying the introduction of the attachment substrate, allowing most to settle naturally, actually improves the efficiency of metamorphosis attachment. In the intermediate rearing stage, adding only high-organic-matter foods such as yeast and algae powder to the feed results in low feeding rates and very slow growth for juveniles. However, adding low-nutrient components such as sea mud significantly improves the feeding rate and growth efficiency of juveniles. Summary of the Invention

[0006] The purpose of this invention is to provide a highly efficient artificial breeding and intermediate rearing method for *Sinocyclocheilus rubescens*. This method optimizes every key step in the *Sinocyclocheilus rubescens* seedling cultivation process, including broodstock maturation and spawning induction, water quality control, pest control, planktonic larval cultivation and attachment, and intermediate rearing of juveniles. This significantly improves the efficiency of artificial breeding and intermediate rearing while reducing production costs. This method can effectively control the artificial breeding time of *Sinocyclocheilus rubescens* and even enable off-season seedling cultivation, overcoming a series of problems such as unstable spawning induction effects, cumbersome operation, damage to broodstock, high costs, difficulty in controlling seedling water quality, frequent pest outbreaks, low larval metamorphosis and attachment rates, and slow juvenile growth. It achieves highly efficient breeding and intermediate rearing of *Sinocyclocheilus rubescens* seedlings and has a very broad application prospect in the field of tropical sea cucumber artificial breeding and aquaculture.

[0007] The first objective of this invention is to provide a method for inducing labor in sea cucumbers with white feet, comprising the following steps:

[0008] The mature gonadal-bound sea cucumbers are first air-dried, then transferred to seawater, where sodium sulfite is added to create oxygen deficiency in the water and stimulate spawning.

[0009] Preferably, after adding sodium sulfite to induce oxygen deficiency in the water and stimulate spawning, the seawater is replaced, and then sperm from sea cucumbers is added for further induction.

[0010] Preferably, the air-drying time is 20-30 minutes. More preferably, it is 30 minutes.

[0011] Preferably, adding 0.1 g / L sodium sulfite to induce oxygen deficiency in the water body and stimulate spawning for 20-30 minutes is preferred. 30 minutes is even more preferred.

[0012] The second objective of this invention is to provide a highly efficient method for artificial breeding and intermediate rearing, including water quality control, broodstock maturation and spawning induction, pest control, planktonic larval cultivation and attachment, and intermediate rearing of juvenile sea cucumbers;

[0013] The aforementioned method for inducing labor in sea cucumbers with white feet is the same as the method used to induce labor in sea cucumbers with white feet mentioned above.

[0014] Preferably, the following steps are included:

[0015] A. Water quality control: The natural seawater used must meet the Class II seawater quality standard and undergo filtration and ultraviolet sterilization before use.

[0016] B. Maturation and Spawning of Sea Cucumbers: Wild sea cucumbers are collected in advance before the breeding season to form a breeding population. The parent sea cucumbers are temporarily housed in a maturation tank within a greenhouse. The water temperature in the maturation tank is higher than in the natural sea area. The sea cucumbers feed on benthic diatoms in the tank, and are simultaneously fed sea mud and Sargassum powder as supplementary feed to promote gonadal maturation. The spawning induction method involves placing the parent sea cucumbers in a basket to air dry for 30 minutes, then transferring them to a plastic bucket filled with filtered seawater. Sodium sulfite is added to create oxygen deficiency in the water, and this stimulation is continued for 20-30 minutes. The seawater is then replaced, and sperm obtained through dissection is used for induction. Spawning occurs 2-4 hours later. After spawning stops, the parent sea cucumbers are removed. The fertilized eggs are filtered through a 100-mesh silk screen to remove large impurities, and then concentrated through a 400-mesh silk screen to remove most of the excess sperm. The fertilized egg density is adjusted to 0.5-0.7 eggs / mL for hatching and larval rearing.

[0017] C. Juvenile cultivation of sea cucumbers: Juvenile sea cucumbers are cultivated in an indoor closed environment. The entire cultivation process is maintained with appropriate aeration so that the juveniles are always in a suspended state. They are fed with a mixture of Chaetoceros algae liquid and baker's yeast suspension in a certain ratio. Depending on the turbidity of the culture water, half of the water is replaced every 5-7 days using a siphon method. Dead juveniles, uneaten feed and feces are removed from the bottom of the tank regularly using a siphon method.

[0018] D. Attachment and intermediate rearing of *Salix rubra* larvae: When most larvae have developed into jar-shaped larvae, reduce aeration appropriately and continue rearing until the number of planktonic larvae in the water significantly decreases. Then, begin introducing polyethylene corrugated sheet attachment substrate at a density of 2-3 frames / m³. 2 ;

[0019] After the sea cucumber larvae attach, their diet is changed to benthic diatoms, marine red yeast, chlorella powder, and sargassum powder. The marine red yeast, chlorella powder, and sargassum powder are mixed in a mass ratio of 1:1:2 and fed once a day. The amount of feed increases from 1 ppm to 5 ppm as the larvae grow. The benthic diatoms are fed once every 3 days, and the amount of feed also increases as the larvae grow. Three hours after feeding, the water quality is kept stable by continuous flow of water. Two weeks after attachment, when the juvenile sea cucumbers reach a length of about 1 mm, an appropriate amount of fresh sea mud is added daily. During the intermediate growth period, the number of daily feedings can be increased to improve the growth rate of the sea cucumbers. Continuous cultivation yields large-sized sea cucumbers.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) This invention uses a maturation tank in a greenhouse for the rearing and maturation of broodstock. The greenhouse roof is transparent, which can effectively increase the temperature of the maturation tank. In addition, artificial feeding is beneficial to the early maturation of the broodstock gonads. The indoor environment is more stable and easier to preserve the gonads of sea cucumbers. It effectively avoids natural ovulation caused by typhoons and rainstorms in the natural sea area. Some sea cucumbers that have been induced to spawn can be induced to spawn again after a period of recovery in the rearing tank.

[0022] (2) Based on the physiological habits of sea cucumbers, this invention uses sodium sulfite to induce hypoxia and asphyxiation, achieving excellent spawning-inducing effects. Compared to existing traditional artificial spawning-inducing methods for tropical sea cucumbers, such as the running water stimulation method and the shade drying method, which have a maximum spawning rate of 13.3-18.0%, this method is significantly more effective (spawning rate as high as 75-81%). Sodium sulfite has no obvious toxicity to parent sea cucumbers and is inexpensive; traditional spawning-inducing methods must be performed at night, while this invention has no time requirement for spawning induction, is highly operable, easy to promote, reduces the amount of parent sea cucumbers, and can provide a stable supply of fertilized eggs for large-scale sea cucumber breeding.

[0023] (3) This invention uses a delayed placement of the attachment substrate to reduce the interference of water flow changes on the larvae. Traditional sea cucumber seedling cultivation involves placing the attachment substrate when the proportion of jar-shaped larvae reaches 30-50%. Based on the ecological habits of the larvae of the white-footed sea cucumber, this invention places the attachment substrate after some larvae have naturally attached to the bottom of the container and their adaptability to the environment has increased. This effectively avoids the mass mortality of larvae during the attachment metamorphosis period caused by sudden environmental changes due to the addition of the attachment substrate.

[0024] (4) This invention uses 100-mesh mesh to cultivate benthic diatoms. Compared with traditional polyvinyl chloride corrugated plates, mesh is cheaper, easier to operate, and has a larger specific surface area, which is conducive to accelerating the attachment and growth of benthic diatoms. The attached benthic diatoms can be easily peeled off, effectively supplementing fresh food for juvenile sea cucumbers.

[0025] (5) This invention uses marine red yeast, chlorella powder, sargassum powder and sea mud as feed for juvenile sea cucumbers. There is little research on the feed for juvenile sea cucumbers. Traditionally, algae powder, benthic diatoms, yeast and probiotics are used as feed during the intermediate growth period of sea cucumbers. The addition of sea mud in this invention is more in line with the feeding habits of sea cucumbers and can significantly accelerate the growth of juvenile sea cucumbers. Attached Figure Description

[0026] Figure 1 The jade-footed sea cucumber parent sea cucumbers are ripened in a ripening pool inside a greenhouse, and the workshop ceiling is light-permeable.

[0027] Figure 2 These are the gonads of mature sea cucumbers with white feet; the left is female and the right is male. Photographed on August 1, 2023.

[0028] Figure 3 The indoor holding tanks for ginseng are designed to prevent light from entering the workshop.

[0029] Figure 4 These are the gonads of ginseng parents in a temporary rearing tank. The gonads can be restored multiple times, and some parents can be preserved for a long time. The left picture was taken on December 8, 2023, and the right picture was taken on March 15, 2024.

[0030] Figure 5 To optimize the spawning process: A. Air-dry for 30 minutes; B. Stimulate with sodium sulfite for 20-30 minutes to induce hypoxia in sea cucumbers; C. Induce hypoxia and asphyxiation in sea cucumbers; D. Dissect sperm to induce spawning.

[0031] Figure 6 To collect fertilized eggs, a 100-mesh sieve is first used to filter out large particles of impurities, and then a 400-mesh sieve is used to collect the eggs while removing excess sperm.

[0032] Figure 7 This shows the changes in the developmental status of the planktonic larvae of the sea cucumber *Sargassum fusiforme* over time. 2d represents the second day after fertilization, and so on.

[0033] Figure 8 The change in body length of the planktonic larvae of the sea cucumber with jade feet over time;

[0034] Figure 9 To cultivate benthic diatoms using corrugated plates and 100-mesh netting, the left image shows the diatom cultivation pond, and the right image shows the benthic diatoms on the netting being rubbed into clean seawater as food for juvenile sea cucumbers.

[0035] Figure 10 After corrugated plate attachment substrate was added, the larvae were swept to the bottom by the water flow, resulting in a large number of deaths.

[0036] Figure 11 During the intermediate growth of juvenile sea cucumbers, benthic diatoms, marine mud, algae powder, and marine red yeast are added.

[0037] Figure 12 The image shows a juvenile sea cucumber six months after attachment, taken on April 11, 2024.

[0038] Figure 13 To compare the changes in the body length of juvenile sea cucumbers with and without sea mud over time, both groups were simultaneously fed benthic diatoms, algal powder, and yeast, in addition to sea mud.

[0039] Figure 14 The left image shows the sea mud added during the intermediate growth process of juvenile sea cucumbers, and the right image shows the sea sand added.

[0040] Figure 15 Experimental sites showcasing the use of sea mud alone, sea sand alone, sea mud + Sargassum powder, and sea sand + Sargassum powder as food for juvenile sea cucumbers. Detailed Implementation

[0041] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the methods and equipment used in the present invention are commonly used methods and equipment in this technical field.

[0042] Example 1: Inducing maturation and spawning of parent sea cucumbers with white feet

[0043] Starting in July 2023, artificial breeding experiments of *Sinonovacula julibrissin* were conducted at the Shenzhen experimental base to further optimize the maturation and spawning conditions of the parent sea cucumbers. The relevant process is as follows:

[0044] Pre-caught sea cucumbers were harvested from the Jiediaosha waters of Daya Bay on July 11 and temporarily held in an indoor ripening tank. Figure 1 At this time, the sea cucumbers' gonads were not yet fully developed. During the temporary rearing period, the sea cucumbers fed on benthic diatoms that grew naturally at the bottom of the tank. They were given a mixture of sea mud and Sargassum powder weekly to enhance their nutrition. A constant flow of water was used to maintain stable water quality, and the water temperature in the tank (28-30℃) was slightly higher than in the natural sea area (27-29℃). Dissection in early August revealed that the gonads of the *Sargassum fusiforme* sea cucumbers were fully mature. Figure 2 Four labor induction experiments were conducted on August 7-8 and August 18-19, 2023 (Table 1).

[0045] 1. Air-drying to induce fertilization: Place the artificially ripened ginseng in a basket and air-dry for 30 minutes. Figure 5 -A), and then transfer it to a plastic bucket filled with filtered seawater;

[0046] 2. Sodium sulfite hypoxia stimulation to induce spawning: Transfer the broodstock to a plastic bucket filled with filtered seawater, add ~0.1g / L sodium sulfite to induce hypoxia in the water, and continue stimulation for 20-30 minutes. Figure 5 -B), then replace with clean seawater;

[0047] 3. Air drying + sodium sulfite hypoxia stimulation to induce labor: Place the ginseng in a basket and air dry for 30 minutes. Figure 5 -A), then transfer to a plastic bucket filled with filtered seawater, add ~0.1g / L sodium sulfite to deoxygenate the water, and continue stimulation for 20-30 minutes ( Figure 5 -B), then replace with clean seawater;

[0048] After the operation of Example 1 of this invention, it was found that the gonads of the parent sea cucumbers were fully mature. Compared with the effects of air drying alone, sodium sulfite hypoxia stimulation, and the combination of the two stimulations, it was found that the effect of air drying + sodium sulfite hypoxia stimulation was better in inducing spawning. A total of 4 spawning induction experiments were conducted, with a spawning success rate as high as 75-81%, and about 20 million fertilized eggs were obtained. The average number of eggs laid by female sea cucumbers was about 2 million per head. The spawning induction rate using this method was much higher than that of air drying alone (0) and sodium sulfite hypoxia alone (43%).

[0049] The oxytocin induction rate (%), the number of fertilized eggs (number), and the average number of eggs laid (number per head) are calculated as follows:

[0050] Induction rate (%) = (Semen ejaculation rate + Ovulation rate) / Induction rate * 100

[0051] Number of fertilized eggs (in cells) = Number of fertilized eggs in a 6-well plate / 5 * (100 or 200 dilution factor) * Volume of water containing fertilized eggs

[0052] Average egg production (eggs / head) = Number of fertilized eggs / Number of eggs laid

[0053] Note: The method for counting fertilized eggs is as follows: after the sea cucumber stops ovulating, remove the parent sea cucumber, first filter out large particles of impurities using a 100-mesh sieve, and then collect the fertilized eggs using a 400-mesh sieve. Figure 6 Pour the fertilized eggs into clean seawater, use a measuring cup to count the volume of water (mL), stir gently to mix evenly, and then use a pipette to transfer 5mL of liquid to a beaker filled with 500mL or 1000mL of clean seawater. Stir evenly, and then repeatedly transfer 5mL of liquid to a 6-well plate to count the fertilized eggs.

[0054] Example 2: Inducing labor in parent sea cucumbers with white feet

[0055] Sea cucumber collection: On September 11, the broodstock sea cucumbers were harvested by diving in the Jiediaosha area of ​​Daya Bay (Daya Bay harvest 1). A typhoon had just occurred a few days before the harvest. Following the method in Example 1, the sea cucumbers were induced to spawn by air drying for 30 minutes and then stimulating them with sodium sulfite for 30 minutes. However, no sperm or eggs were released. Dissection revealed that the gonads of the broodstock sea cucumbers had completely degenerated (Table 1).

[0056] Example 3: Inducing maturation and spawning of parent sea cucumbers with white feet

[0057] Sea cucumber collection: On September 17, adult sea cucumbers were harvested by diving from Dapeng Bay, Shenzhen (Dapeng Bay harvest 2). This sea area was less affected by typhoons. At this time, about 50% of the sea cucumbers had full gonads. The sea cucumbers were temporarily kept in indoor tanks with normal aeration. The sea cucumbers mainly fed on benthic diatoms and sediment that grew naturally at the bottom of the tank. From September 21 to 23, 2023, the sea cucumbers were induced to spawn (Table 1).

[0058] Air drying + sodium sulfite hypoxia stimulation to induce spawning: Place the adult sea cucumber in a basket to air dry for 30 minutes, then transfer it to a plastic bucket filled with filtered seawater, add sodium sulfite to make the water hypoxic, and continue to stimulate for 30 minutes, then replace with clean seawater.

[0059] Through the operation of Example 3 of the present invention, it was found that the combination of air drying and sodium sulfite hypoxia stimulation had a good effect on inducing spawning in naturally matured sea cucumber parents in the wild. A total of 3 spawning induction experiments were conducted, with a success rate of 2, and approximately 4.7 million fertilized eggs were obtained.

[0060] Example 4: Induction of labor in parent sea cucumbers with white feet

[0061] Sea cucumber collection: Sea cucumbers were harvested by diving from Dapeng Bay, Shenzhen on September 22 (Dapeng Bay harvest 3). At this time, the sea cucumbers had full gonads. The sea cucumbers were temporarily kept in indoor tanks with normal aeration. The sea cucumbers mainly fed on benthic diatoms and sediment that grew naturally at the bottom of the tank. Spawning was induced on September 24-25, 2023. After induced spawning, the sea cucumbers were temporarily kept for a period of time and then induced to spawn again (Table 1).

[0062] Air drying + sodium sulfite hypoxia stimulation + sperm induction to induce spawning: The adult sea cucumbers were placed in a basket and air-dried for 30 minutes. ~0.1 g / L sodium sulfite was added to induce hypoxia in the water, and this stimulation was continued for 30 minutes. Figure 5 -B), then the seawater was changed, and the male gonads obtained by dissection were crushed in clean seawater using a 300-mesh sieve to obtain sperm for induction ( Figure 5 -D);

[0063] Air-drying + sperm-induced labor induction: After air-drying the adult sea cucumber in a basket for 30 minutes, put it into a bucket filled with clean seawater and add sperm obtained by dissection to induce labor. The amount of sperm added should be such that the water is slightly milky white.

[0064] Air-drying + temperature stimulation + sperm induction to induce labor: Place the adult sea cucumber in a basket and air-dry for 30 minutes. Transfer it to a bucket filled with clean seawater and slowly add hot fresh water while stirring. Increase the water temperature from 25℃ to 30℃ and decrease the salinity from 31 to 29. Add sperm obtained by dissection to induce labor. The amount of sperm added should be such that the water turns slightly milky white.

[0065] Parent sea cucumber temporary rearing and recovery: After induced spawning, the parent sea cucumbers were returned to the temporary rearing tank to recover for a period of time. During this period, the parent sea cucumbers were raised in the same environment and fed as above. After 3, 4 and 7 weeks of temporary rearing, they were induced to spawn by air drying for 30 minutes, sodium sulfite hypoxia stimulation for 30 minutes and sperm induction.

[0066] Through the operation of Example 4 of this invention, compared with the traditional air-drying method, temperature stimulation method, and sperm induction method, air-drying + sodium sulfite hypoxia stimulation + sperm induction can observe oviposition behavior within 2-4 hours of induced spawning, with a greater number of eggs laid. Furthermore, even when the water temperature drops to 24-25℃ in November, induced spawning can still be successfully induced. A total of 5 induced spawning experiments were conducted, with a success rate of 100%, far exceeding the ordinary air-drying and flowing water stimulation methods. Approximately 25 million fertilized eggs were obtained, with a gamete fertilization rate of over 95%. The vast majority of fertilized eggs developed normally. The gonads of the parent sea cucumbers can be preserved long-term in indoor tanks. Dissection in December revealed that the gonads of some female parent sea cucumbers were still full. Figure 4 ).

[0067] Table 1. Comparison of labor-inducing effects of broodstock sea cucumber.

[0068]

[0069] Note: The parent fish matured in the indoor pond were caught by diving in the Jiediaosha waters of Daya Bay on July 11; the parent fish caught in Daya Bay 1 were caught by diving in the Jiediaosha waters of Daya Bay on September 11; the parent fish caught in Dapeng Bay 2 were caught by diving in the Dapeng Bay waters of Shenzhen on September 17; the parent fish caught in Dapeng Bay 3 were caught by diving in the Dapeng Bay waters of Shenzhen on September 22; the parent fish temporarily held in the indoor pond were the parent fish caught in Daya Bay 2 and Dapeng Bay 3, which were temporarily held in the greenhouse pond. Figure 3 When using sodium sulfite for hypoxic stimulation, DO < 0.5 mg / L -1 ;- indicates that it was not counted.

[0070] Example 5: Inducing spawning in brown-ringed sea cucumber using an optimized method

[0071] *Holothuria fuscocinerea*, belonging to the genus *Holothuria* of the family Holothuridae, is an important tropical sea cucumber. In the Daya Bay waters of Shenzhen, *Holothuria fuscocinerea* is a common species, typically inhabiting the bottom of corals or reefs, feeding on detritus and sediment. It has been reported that *Holothuria fuscocinerea* has not yet been successfully bred artificially, primarily due to the lack of effective methods for inducing spawning. This species is highly sensitive to environmental changes, and traditional air-drying methods easily damage the sea cucumber's skin, resulting in extremely poor spawning induction; no successful cases of spawning induction have been reported. Artificial spawning induction experiments for *Holothuria fuscocinerea* were conducted at the Shenzhen Experimental Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, in June-July 2024.

[0072] Sea cucumber harvesting and temporary holding: Sea cucumbers were harvested in Daya Bay on June 1, 2024 and temporarily held in indoor culture ponds. During this period, they were fed sea mud and Sargassum powder and the water quality was kept stable by continuous flow of water.

[0073] Induced spawning in parent sea cucumbers: On June 24, 2024, spawning was induced using a combination of half an hour of air drying, half an hour of sodium sulfite stimulation, and sperm induction. The specific method was as follows: parent sea cucumbers were placed in a basket to air dry for 30 minutes, then 0.1 g / L sodium sulfite was added to create oxygen deficiency in the water, and this stimulation was continued for 30 minutes. Afterward, the seawater was replaced, and sperm obtained through dissection was used for induction. A total of 30 sea cucumbers were induced to spawn, with 4 producing sperm but no eggs. After air drying, the sea cucumbers' skin was easily damaged. Upon re-entry into the water, it was found that the sea cucumbers had absorbed air, and most individuals floated on the surface. Therefore, we hypothesize that air drying is not conducive to induced spawning in brown-ringed sea cucumbers. On June 25, 2024, sodium sulfite stimulation for half an hour was used, followed by sperm induction to induce spawning (the parent sea cucumbers were transferred to a plastic bucket filled with filtered seawater, and ~0.1g / L sodium sulfite was added to make the water hypoxic, and the stimulation was continued for 30 minutes. After that, clean seawater was replaced, and sperm obtained by dissection was added to induce spawning. The amount of sperm added was such that the water was slightly milky white). There were 25 sea cucumbers in the same batch. Among them, 5 sea cucumbers released sperm and 4 sea cucumbers released eggs, and a total of 1.6 million fertilized eggs were obtained, with a fertilization rate of nearly 100%. The larvae developed normally. After two weeks of temporary rearing, spawning was induced again. On July 9, 2024, sodium sulfite stimulation for half an hour was used, followed by sperm induction to induce spawning (the parent sea cucumbers were transferred to a plastic bucket filled with filtered seawater, and ~0.1g / L sodium sulfite was added to deoxygenate the water for 30 minutes, followed by replacement with clean seawater and the addition of sperm obtained through dissection to induce spawning). Of the 20 sea cucumbers in the same batch, 6 released sperm but did not ovulate. On July 10, 2024, sodium sulfite stimulation for half an hour was used again, followed by sperm induction to induce spawning (the parent sea cucumbers were transferred to a plastic bucket filled with filtered seawater, and ~0.1g / L sodium sulfite was added to deoxygenate the water for 30 minutes, followed by replacement with clean seawater and the addition of sperm obtained through dissection to induce spawning). Of the 18 sea cucumbers in the same batch, 3 released sperm but did not ovulate. This method has a certain oxytocin-inducing effect on brown-ringed sea cucumbers. Dissection of the sea cucumbers revealed that the color of the female gonads gradually faded, suggesting that the gonads of brown-ringed sea cucumbers cannot be preserved for a long time in an indoor environment. Therefore, considering the characteristics of brown-ringed sea cucumbers (easily damaged epidermis), the oxytocin-inducing method for jade-footed sea cucumbers was improved (only sodium sulfite hypoxia stimulation + sperm induction was retained), which can induce oxytocin production in this difficult-to-induce sea cucumber, achieving a breakthrough from zero.

[0074] Example 6: High-efficiency artificial cultivation of planktonic larvae of the sea cucumber *Sinonovacula julibrissin*

[0075] Artificial breeding experiments of *Salvia miltiorrhiza* were conducted at the Shenzhen Experimental Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, from September to November 2023. The relevant process is as follows:

[0076] Water quality control: The natural seawater used for seedling cultivation was taken from the sea area near the experimental station. Testing showed that the levels of all pollutants were below the Class II seawater quality standard. The extracted seawater was filtered through sand and then further filtered and disinfected with ultraviolet light using a 5-micron pore size filter bag. During this experiment, the salinity of the treated seawater ranged from 28 to 33, and the temperature ranged from 28 to 31°C.

[0077] Induced spawning in *Solanum tuberosum*: The parent sea cucumbers were harvested from Dapeng Bay, Shenzhen. However, due to typhoon impact, dissection revealed that 50% of the parent sea cucumbers had degenerated gonads, while the remaining individuals had very full gonads. After 4 days of temporary rearing, spawning was induced on September 21, 2023: The sea cucumbers were first removed from the rearing tank and air-dried for 30 minutes. Then, they were transferred to a plastic bucket filled with filtered seawater. Sodium sulfite (0.1 g / L) was added to create oxygen deficiency in the water, and this stimulation was continued for 30 minutes. The seawater was then replaced, and sperm obtained through dissection was used for induction. The amount of sperm added was adjusted until the water turned slightly milky white. Three hours later, a large amount of sperm was observed being released, followed by ovulation. After the sperm and ovulation process was completed, fertilized eggs were collected. Large particles were first filtered through a 100-mesh sieve, and then the fertilized eggs were collected through a 400-mesh sieve. Figure 6 At the same time, excess sperm are removed, and then the fertilized eggs are placed in several 500L tanks for incubation at a density of 0.5-0.7 eggs / mL. Adequate light has no adverse effect on the planktonic larvae, but direct sunlight should be avoided, and the fertilized eggs should be continuously aerated to keep them suspended.

[0078] Cultivation method of planktonic larvae of sea cucumber with white feet: On the second day, the planktonic larvae of sea cucumber develop into the small ear larvae stage. At this time, the larvae have started to feed. During the cultivation of planktonic larvae, depending on the turbidity of the water, change 1 / 2 of the water every 5-7 days. Stop aeration when changing the water. Most of the larvae will swim to the surface of the water. Use the siphon method to suck the water from the bottom of the bucket. During this period, shake the siphon tube up and down appropriately to reduce damage to the larvae.

[0079] Feeding began immediately upon observation of the small-eared larvae. Before the larvae developed into late-middle-eared larvae, each feeding consisted of 2 L / 1000L of Chaetoceros algae and 2 L / 3000L of baker's yeast suspension, twice a day, in the morning and afternoon. The feeding was observed 3-4 hours after feeding, and the amount was adjusted accordingly. From the large-eared larvae to the jar-shaped larvae stage, the feeding amount increased to 3 L / 1000L of Chaetoceros algae and 1 L / 1000L of baker's yeast suspension. Subsequently, as the larvae in the water metamorphosed and attached themselves, the feeding amount was gradually reduced. Finally, approximately 16 days after fertilization, the five-tentacle larvae appeared. Figure 7 , Figure 8 Our long-term practice has shown that when the density of planktonic larvae in water is high, the introduction of attachment substrates will cause a large number of larvae to sink to the bottom and die. Figure 10Therefore, when most of the planktonic larvae have metamorphosed into jar-shaped larvae, reduce aeration appropriately and continue cultivation until the larval density in the water decreases significantly. Then, begin adding polyethylene corrugated sheet attachment substrates at a quantity of 2-3 frames / m². 2 This effectively reduces larval mortality. The corrugated board is disinfected and coated with diatoms before use—diatoms must be applied at least one week in advance to improve the adhesion effect. The larger side of the corrugated board is placed against the bottom of the container; later, the juveniles at the bottom will disperse onto the substrate on their own.

[0080] Overfeeding increases impurities at the bottom of the container and suspended particles in the water, inhibiting the development of larvae. Therefore, the amount of feed should be moderate each time, and it is advisable to feed in small amounts multiple times. Larvae are cultivated using Chaetoceros muelleri and baker's yeast as feed, with the density of Chaetoceros muelleri not less than 2 million cells / mL. Before feeding, the feed should be examined under a microscope to check for contamination by protozoa. Normal algae water is dark and bright in color, with no obvious odor. Avoid feeding old or spoiled feed. If there are too many impurities at the bottom of the container, the bottom should be slurred in time to remove dead larvae, uneaten feed, and feces.

[0081] Intermediate rearing of the jade-footed sea cucumber: After the corrugated plate is deployed, the feed is changed to benthic diatoms, marine red yeast, chlorella powder, sargassum powder, and sea mud. Figure 11 The initial feed for sea cucumbers is a mixture of marine red yeast, Chlorella powder, and Sargassum powder in a 1:1:2 ratio. The algae powder is crushed using a 160-mesh sieve and then dissolved in clean seawater, stirred thoroughly, and used once daily. The feeding amount increases from 1 ppm to 5 ppm as the fry grow. Benthic diatom flushing solution is given every 3 days. Figure 9 The amount of feed also increases as the seedlings grow. Two weeks after the sea cucumbers attach, sea mud is added. The sea mud used is the floating mud from the intertidal zone at low tide. Large particles are filtered out using a sieve, and a layer of bottom mud is evenly sprinkled onto the bottom of the breeding tank every day to cover it. Figure 14 Three hours after feeding, maintain stable water quality using a continuous flow of water. During the intermediate rearing period, the daily feeding frequency can be increased to improve the growth rate of sea cucumbers, and continuous cultivation can yield large-sized sea cucumber seedlings. Figure 12 The growth rates of juvenile sea cucumbers fed with and without sea mud were compared. The group without sea mud was fed the same benthic diatoms, algal powder, and yeast as the group with sea mud, but their growth rate was significantly slower. Figure 13 );

[0082] On April 12, 2024, a food experiment was conducted on juvenile sea cucumbers, using sea mud and sea sand. Figure 14 , Figure 15Sea mud + Sargassum powder and sea sand + Sargassum powder were used as feed for juvenile sea cucumbers. Seven juvenile sea cucumbers were placed in each bucket, with three replicates per group. They were raised for one month, with the water changed once a week, replacing half of the water each time. Feed was added only at the beginning and not afterward. After the experiment, the weight and survival rate of the sea cucumbers were measured. It was found that the juvenile sea cucumbers in the sea mud + Sargassum powder and sea sand + Sargassum powder groups had better growth, while the effects of using sea mud or sea sand alone were not good (Table 2).

[0083] Table 2

[0084]

[0085] Note: For the same row, different superscript letters indicate that there are significant differences between the data groups (P<0.05).

[0086] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for inducing labor in sea cucumber with white feet, characterized in that, Includes the following steps: The mature gonadal-bound sea cucumbers are first air-dried, then transferred to seawater, where sodium sulfite is added to create oxygen deficiency in the water and stimulate spawning.

2. The labor-inducing method according to claim 1, characterized in that, After adding sodium sulfite to deoxygenate the water and stimulate spawning, the seawater is replaced, and then sperm from sea cucumbers is added to induce spawning.

3. The labor-inducing method according to claim 1, characterized in that, The air-drying time is 20-30 minutes.

4. The labor-inducing method according to claim 3, characterized in that, The air-drying time is 30 minutes.

5. The labor-inducing method according to claim 1, characterized in that, Adding 0.1g / L sodium sulfite to deoxygenate the water body stimulates spawning for 20-30 minutes.

6. The labor-inducing method according to claim 5, characterized in that, Stimulate labor for 30 minutes.

7. A method for efficient artificial breeding and intermediate rearing, characterized in that, This includes water quality control, broodstock maturation and spawning promotion, pest control, planktonic larval cultivation and attachment, and intermediate growth of juvenile sea cucumbers; The induced labor of the parent sea cucumber is performed using the induced labor method for the jade-footed sea cucumber described in claim 1.

8. The method according to claim 7, characterized in that, Specifically, the following steps are included: A. Water quality control: The natural seawater used must meet the Class II seawater quality standard and undergo filtration and ultraviolet sterilization before use. B. Maturation and Spawning of Sea Cucumbers: Wild sea cucumbers are collected in advance before the breeding season to form a breeding population. The parent sea cucumbers are temporarily raised in a maturation tank in a greenhouse. The water temperature in the maturation tank is higher than that in the natural sea area. The sea cucumbers feed on benthic diatoms in the tank and are fed with sea mud and Sargassum powder as supplementary feed to promote the maturation of the sea cucumber gonads. The spawning induction method is to place the parent sea cucumbers in a basket to air dry for 30 minutes, and then transfer them to a plastic bucket filled with filtered seawater. Sodium sulfite is added to make the water hypoxic and continuously stimulated for 20-30 minutes. After that, the seawater is changed, and sperm obtained by dissection is used for induction. The parent sea cucumbers spawn after 2-4 hours. After the parent sea cucumbers stop spawning, they are removed. The fertilized eggs are filtered through a 100-mesh silk screen to remove large impurities, and then concentrated through a 400-mesh silk screen to remove most of the excess sperm. The density of fertilized eggs is adjusted to 0.5-0.7 eggs / mL for hatching and larval rearing. C. Juvenile cultivation of sea cucumbers: Juvenile sea cucumbers are cultivated in an indoor closed environment. The entire cultivation process is maintained with appropriate aeration so that the juveniles are always in a suspended state. They are fed with a mixture of Chaetoceros algae liquid and baker's yeast suspension in a certain ratio. Depending on the turbidity of the culture water, half of the water is replaced every 5-7 days using a siphon method. Dead juveniles, uneaten feed and feces are removed from the bottom of the tank regularly using a siphon method. D. Attachment and intermediate rearing of *Salvia miltiorrhiza* larvae: When most larvae have developed into jar-shaped larvae, reduce aeration appropriately and continue rearing until the number of planktonic larvae in the water significantly decreases. Then, begin introducing polyethylene corrugated sheet attachment substrate at a density of 2-3 frames / m³. 2 ; After the sea cucumber larvae attach, their diet is changed to benthic diatoms, marine red yeast, chlorella powder, and sargassum powder. The marine red yeast, chlorella powder, and sargassum powder are mixed in a mass ratio of 1:1:2 and fed once a day. The amount of feed increases from 1 ppm to 5 ppm as the larvae grow. The benthic diatoms are fed once every 3 days, and the amount of feed also increases as the larvae grow. Three hours after feeding, the water quality is kept stable by continuous flow of water. Two weeks after attachment, when the juvenile sea cucumbers reach a length of 1 mm, an appropriate amount of fresh sea mud is added daily. During the intermediate growth period, the daily feeding frequency can be increased to improve the growth rate of the sea cucumbers. Continuous cultivation yields large-sized sea cucumbers.

Images