A method for off-season cultivation of scallop seedlings

Abstract

This application relates to the field of bio-agricultural breeding technology, and in particular to a method for off-season scallop seedling cultivation and its application. This application utilizes an indoor low-temperature circulating water system and employs a comprehensive environmental and nutritional control strategy involving "artificial low-temperature dormancy – gradient warming for maturation – off-season induced spawning – low-temperature acclimatization transition" to disrupt the natural seasonal rhythm of scallop reproduction. This not only ensures the survival rate of scallops released into the sea but also allows for staggered release times, shortens the cultivation cycle, and reduces summer mortality.

Description

Technical Field

[0001] This application relates to the field of bio-agricultural breeding technology, specifically to a method for off-season seedling cultivation of Yesso scallops and its application. Background Technology

[0002] Ezo scallop ( Patinopecten yessoensis As a cold-water bivalve with high economic value, it is mainly distributed in northern China, Hokkaido, Japan, and the Far East waters of Russia. Its growth and development exhibit a significant seasonal rhythm, with the natural breeding season typically concentrated in spring (water temperature 8℃-12℃). In traditional aquaculture models, the supply of seedlings is strictly limited by the natural seasons, resulting in a fixed aquaculture cycle and a concentrated market entry time, which easily leads to market price fluctuations.

[0003] Traditional seasonal seedling cultivation faces severe challenges, such as: 1) "Summer mortality" syndrome: In the traditional model, seedlings hatch in May-June. By July-August, when temperatures reach 23℃-25℃, the juveniles are still small, with a large surface area to volume ratio and a high metabolic rate, making them extremely vulnerable to environmental stress. 2) Growth cycle and market bottleneck: The growth cycle of seasonal seedlings is entirely dependent on the natural season. Due to the late start, they cannot reach the ideal overwintering size before winter, resulting in a prolonged cultivation cycle. In addition, because the entire industry operates at the same pace, the market entry time of adult scallops is highly concentrated, which can easily cause drastic fluctuations in market prices due to oversupply, reducing the economic benefits for farmers. 3) Instability of traditional seedling cultivation: Relying on seed collection from natural sea areas or simple indoor seedling cultivation, it is greatly affected by spring cold waves, sandstorms, and fluctuations in nearshore water quality. The instability of natural water temperature often leads to asynchronous gonadal development in parent scallops, resulting in inconsistent spawning quality. In view of this, this application is hereby submitted. Summary of the Invention

[0004] To address the aforementioned technical problems, this application proposes a novel method for cultivating off-season scallop seedlings. Specifically, the technical solution of this application is as follows: This application first provides a method for cultivating off-season scallop seedlings, including the following steps: 1) Parent shell screening; 2) Gradual heating to promote ripening; 3) Off-season induction of spawning, fertilization, and hatching; 4) Larval rearing and metamorphosis; 5) Juvenile shellfish cultivation; 6) Domestication before entering the sea.

[0005] Furthermore, step 1) of the parent shell selection includes a low-temperature dormancy process: the parent shell culture water temperature is controlled at 3℃-4℃ for 3-5 days; a low-light environment of <100 Lux is maintained to simulate the environment under ice in deep winter; preferably, step 1) of the parent shell selection specifically includes the following steps: selecting healthy individuals with a shell height >10cm, aged 2-3 years, with regular shape, no damage, and robust development; after cleaning the shell surface attachments, they are temporarily raised in a pool at a density of 20-30 individuals / m². 3 ; Maintain the water temperature at 3℃-4℃ for 3-5 days; keep the environment in complete darkness (<100 Lux) to simulate the environment under ice in deep winter. Based on water quality monitoring results and parent shellfish excretion, a small amount of diatoms were fed, and bottom sludge was removed daily.

[0006] Furthermore, step 2) gradient temperature increase for ripening includes the following steps: increasing the temperature at a rate of 0.2-0.5℃ / day, stabilizing the temperature for 3-5 days when the temperature reaches 5℃, 6℃, and 7℃ respectively, and when the water temperature reaches 8℃ and the feeding amount is significantly increased, then synchronizing the ripening process with constant temperature.

[0007] In some aspects, during step 2) of gradient temperature ripening, the photoperiod is controlled at 14L:10D and the light intensity is controlled at 500-800 Lux. In some aspects, during step 2) of gradient heating for ripening, natural feed and substitute feed are provided; preferably, the natural feed includes *Phaeodactylum tricornutum*, *Rhizophora spp.*, *Golden Algae*, and *Platycodon grandiflorus*, and the substitute feed includes vitamin E, emulsified fish oil, and marine red yeast.

[0008] Furthermore, in step 3) off-season spawning and fertilization hatching, the off-season spawning is induced by a combination of "air drying + heat shock"; the combination of "air drying + heat shock" involves air drying the parent scallops for 1-2 hours and then placing them in warm seawater that is 3-4°C (i.e., 11-12°C) higher than the temporary rearing water temperature.

[0009] Preferably, step 3) inducing off-season spawning and fertilization includes the following steps: when the Group Gonadal Index (GSI) is >15%, the female gonads are deep orange-red and the male gonads are milky white, off-season induction is carried out; the parent shellfish are air-dried for about 1-2 hours and placed in seawater that is 3-4℃ higher than the temporary rearing water temperature (i.e., 11℃-12℃); when the number of eggs laid reaches 40-50 / mL, the parent shellfish are removed and examined under a microscope to control 3-5 sperm cells around each egg; the mixed sperm are evenly sprinkled and stirred once every half hour.

[0010] Furthermore, step 4) larval cultivation and metamorphosis includes larval selection and seedling collection and metamorphosis induction; In some aspects, the larval selection steps include: Using the floating habit of D-type larvae of Yesso scallops, siphon selection was carried out to remove dead eggs and deformed individuals from the bottom layer. After selection, the culture density of D-type larvae was quantitatively adjusted to 10-15 larvae / mL per tank. From the late shell apex stage to the eyepoint larval stage, the density was gradually diluted to 2-3 larvae / mL to avoid density stress. Preferably, during the larval selection process, a gradient temperature increase is adopted, with the water temperature slowly raised from 12℃ at hatching time at a rate of 0.5℃ / 3 days, eventually maintaining at 15℃, which promotes metabolism while inhibiting diseases; the water quality is maintained at a salinity of 30-32‰ and a pH of 8.0-8.2; the water exchange rate increases from 20% to 50% with age. In a further preferred embodiment, the larval rearing process adopts a feeding strategy of "golden algae as the base and diatoms to promote growth": in the early stage of larval rearing, golden algae are the main feed to ensure the palatability of the initial feed; in the later stage of larval rearing, diatoms and flat algae are added.

[0011] In some aspects, the seedling collection and metamorphic attachment induction include: When 20-30% of the larvae in the group show eyespots and their legs are active, the seedling collection period begins. Pre-treated attachment substrates are then introduced, and the light is adjusted to weak diffused light of <100 Lux to induce larval attachment metamorphosis by utilizing tactile attraction and photophobia.

[0012] Furthermore, step 5) of juvenile clam cultivation includes the following steps: In the early stages after the larvae are placed on the attachment substrate, the aeration rate is reduced to achieve a micro-aeration state. After microscopic examination confirms that the larvae have completely attached and metamorphosed into juveniles, the aeration rate is increased. As the metabolism of the juveniles increases, the daily water exchange rate is gradually increased to maintain the water exchange rate at more than 2 / 3. Diatoms and flat algae are fed, and the feeding amount is adjusted according to the dynamic gradient of the juveniles' growth stages. In some aspects, the dynamic gradient adjustment of feeding amount is as follows: in the early stage of juvenile growth, the feeding amount is controlled at a maximum of 4×104 cells / mL to ensure the feeding matching degree of the newly metamorphosed juveniles; in the later stage of juvenile growth, as the juveniles' water filtration capacity is enhanced, the feeding amount is gradually increased to a maximum of 8×104 cells / mL to meet the energy requirements for rapid mineralization of the juvenile shell and growth of the soft body.

[0013] Furthermore, step 6) pre-sea acclimatization includes acclimatizing the indoor water temperature, salinity, and light exposure; In some aspects, the acclimatization includes: slowly cooling the water at a rate of 1.0°C per day, gradually reducing the water temperature from the cultivation temperature to the same temperature in the sea area; controlling the salinity difference between the nursery pond and the target sea area to be ≤2‰; gradually increasing the light intensity to simulate natural diffused light of 500-1500 Lux; more preferably, during the acclimatization period, adding concentrated algal liquid rich in highly unsaturated fatty acids, as well as vitamin C and immunopolysaccharides to the feed.

[0014] In some aspects, step 6) pre-sea domestication also includes temporary rearing at sea: when the average shell height of the juvenile oysters reaches 500-800μm or more, the gill filaments are fully developed under a microscope, the foot movements are strong, the mortality rate of the population is less than 1%, and there are few miscellaneous algae on the substrate, temporary rearing at sea is carried out; the temporary rearing at sea is carried out as follows: after the seedlings arrive at the sea area, the seedling bags are first suspended in the middle layer of the inner bay or floating raft with small waves and stable currents at a water depth of 3-5 meters for 3-7 days. After the juvenile oysters have fully adapted to the sea environment, they are then transferred to standard culture cages for normal growth management.

[0015] The advantages of this application include at least the following aspects: This application is based on the synergistic regulation of the entire process of "artificial low-temperature dormancy - gradient warming to promote maturity - off-season induced spawning - low-temperature acclimatization transition," combined with multi-parameter coupling. Before the seedlings are released into the sea, multi-dimensional acclimatization with temperature, salinity, light, and nutrition is carried out to ultimately eliminate the stress response caused by the difference between indoor and outdoor environments, ensuring a high survival rate. At the same time, this application shortens the breeding cycle. Compared with traditional seedlings, off-season seedlings are equivalent to getting a head start of 2-3 months, and the adult oyster growth period can be shortened by more than 2 months, achieving the goal of reaching the target market in the same year or being launched on the market earlier in the following spring.

[0016] This application achieves reverse-time regulation, simulating a spring environment through an indoor circulating water system while the outside world is still in the depths of winter, thus initiating the reproductive process of parent shellfish ahead of schedule. This staggered release into the sea allows seedlings to reach a relatively large size (shell height > 500 μm) by March or April, when the water temperature in the natural sea area rises to a suitable level for growth. Taking advantage of the abundant phytoplankton growth in spring, they complete "explosive growth" before the summer heat arrives, reaching a juvenile size capable of withstanding high temperatures by July. Simultaneously, it avoids the risks of high temperatures, advancing the most vulnerable stage for juvenile shellfish from the hot summer to the cool spring, significantly reducing summer mortality. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1After ripening, female oyster gonad tissue sections were collected from the six groups of processes. Among them, Figure A represents the female oyster gonad tissue section obtained from experimental group 1, Figure B represents the female oyster gonad tissue section obtained from experimental group 2, Figure C represents the female oyster gonad tissue section obtained from experimental group 3, Figure D represents the female oyster gonad tissue section obtained from experimental group 4, Figure E represents the female oyster gonad tissue section obtained from experimental group 5, and Figure F represents the female oyster gonad tissue section obtained from experimental group 6.

[0019] Figure 2 After the six groups of processes were used to promote maturity, male oyster gonad tissue sections were collected. Among them, Figure A represents the male oyster gonad tissue section obtained from experimental group 1, Figure B represents the male oyster gonad tissue section obtained from experimental group 2, Figure C represents the male oyster gonad tissue section obtained from experimental group 3, Figure D represents the male oyster gonad tissue section obtained from experimental group 4, Figure E represents the male oyster gonad tissue section obtained from experimental group 5, and Figure F represents the male oyster gonad tissue section obtained from experimental group 6. Detailed Implementation

[0020] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply.

[0021] The following basic terms or definitions are provided merely to aid in understanding this application. These definitions should not be construed as having a scope less than that understood by those skilled in the art. Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this application are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are still set forth to better explain this application.

[0022] As used in this application, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0023] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0024] The terms "approximately" and "generally" in this application refer to an accuracy range that, as would be understood by those skilled in the art, still guarantees the technical effects of the mentioned features. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0025] Furthermore, the terms first, second, third, (a), (b), (c), and similar terms used in the specification and claims are for distinguishing similar elements and are not necessary for the order of description or chronological sequence. It should be understood that such terms are interchangeable in appropriate contexts, and the embodiments described herein can be implemented in a different order than that described or illustrated herein.

[0026] The method for cultivating off-season scallop seedlings in this application basically includes the following steps: 1) Selection of parent shellfish; 2) Gradual heating to promote maturation; 3) Off-season induction of spawning and fertilization and hatching; 4) Larval cultivation and metamorphosis attachment; 5) Juvenile shellfish cultivation; 6) Pre-sea domestication.

[0027] In some aspects, step 1) of broodstock selection includes a low-temperature dormancy process: the broodstock culture water temperature is controlled at 3℃-4℃ for 3-5 days; a low-light environment of <100 Lux is maintained to simulate the sub-ice environment of deep winter; in some embodiments, step 1) of broodstock selection specifically includes the following steps: selecting healthy individuals with a shell height >10cm, aged 2-3 years, with regular shape, no damage, and robust development; after cleaning the shell surface attachments, they are temporarily raised in a pool at a density of 20-30 individuals / m². 3 ; Maintain the water temperature at 3℃-4℃ for 3-5 days; keep the environment in complete darkness (<100 Lux) to simulate the environment under ice in deep winter. Based on water quality monitoring results and parent shellfish excretion, a small amount of diatoms were fed, and bottom sludge was removed daily.

[0028] In some respects, step 2) gradient temperature increase for ripening includes the following steps: increasing the temperature at a rate of 0.2-0.5℃ / day, stabilizing the temperature for 3-5 days when the temperature reaches 5℃, 6℃, and 7℃ respectively, and when the water temperature reaches 8℃ and the feeding amount is significantly increased, then synchronizing the ripening process with constant temperature.

[0029] In some embodiments, during step 2) gradient temperature ripening, the photoperiod is controlled at 14L:10D and the light intensity is controlled at 500-800 Lux. In some embodiments, during step 2) gradient heating for ripening, natural feed and substitute feed are fed during the ripening period; preferably, the natural feed includes *Phaeodactylum tricornutum*, *Rhizophora spp.*, *Golden Algae*, and *Platycodon grandiflorus*, and the substitute feed includes vitamin E, emulsified fish oil, and marine red yeast.

[0030] In some aspects, in step 3) off-season spawning and fertilization hatching, the off-season spawning is induced by a combination of "air drying + heat shock"; the "air drying + heat shock" combination induction involves air drying the parent scallops for 1-2 hours and then placing them in warmed seawater that is 3-4°C (i.e., 11-12°C) higher than the temporary rearing water temperature.

[0031] In some embodiments, step 3) off-season induction of spawning and fertilization hatching specifically includes the following steps: when the Group Gonadal Index (GSI) > 15%, the female gonads are deep orange-red and the male gonads are milky white, off-season induction is carried out; the parent shellfish are air-dried for about 1-2 hours and placed in warmed seawater that is 3-4°C higher than the temporary rearing water temperature (i.e., 11°C-12°C); when the number of eggs laid reaches 40-50 per mL, the parent shellfish are removed and examined under a microscope to control 3-5 sperm cells around each egg; the mixed sperm are evenly sprinkled and stirred once every half hour.

[0032] In some respects, step 4) larval rearing and metamorphosis includes larval selection and seedling collection and metamorphosis induction; In some embodiments, the larval selection step includes: Using the floating habit of D-type larvae of Yesso scallops, siphon selection was carried out to remove dead eggs and deformed individuals from the bottom layer. After selection, the culture density of D-type larvae was quantitatively adjusted to 10-15 larvae / mL per tank. From the late shell apex stage to the eyepoint larval stage, the density was gradually diluted to 2-3 larvae / mL to avoid density stress. In some preferred embodiments, during the larval selection process, a gradient temperature increase is adopted, with the water temperature slowly raised from 12°C at hatching time at a rate of 0.5°C / 3 days, eventually maintained at 15°C, to promote metabolism while inhibiting disease; the water quality is maintained at a salinity of 30-32‰ and a pH of 8.0-8.2; the water exchange rate increases from 20% to 50% with age. In some preferred embodiments, the larval rearing process adopts a feeding strategy of "golden algae as the base and diatoms to promote growth": in the early stage of larval rearing, golden algae are the main feed to ensure the palatability of the initial feed; in the later stage of larval rearing, diatoms and flat algae are added.

[0033] In some preferred embodiments, the seedling collection and metamorphic attachment induction include: When 20-30% of the larvae in the group show eyespots and their legs are active, the seedling collection period begins. Pre-treated attachment substrates are then introduced, and the light is adjusted to weak diffused light of <100 Lux to induce larval attachment metamorphosis by utilizing tactile attraction and photophobia.

[0034] In some respects, step 5) of juvenile oyster rearing includes the following steps: In the early stages after the larvae are placed on the attachment substrate, the aeration rate is reduced to achieve a micro-aeration state. After microscopic examination confirms that the larvae have completely attached and metamorphosed into juveniles, the aeration rate is increased. As the metabolism of the juveniles increases, the daily water exchange rate is gradually increased to maintain the water exchange rate at more than 2 / 3. Diatoms and flat algae are fed, and the feeding amount is adjusted according to the dynamic gradient of the juveniles' growth stages. In some embodiments, the dynamic gradient adjustment of feeding amount is as follows: during the early stage of juvenile oyster growth, the feeding amount is controlled at a maximum of 4×10⁻⁶. 4 The feeding rate was set at 8 × 10⁴ cells / mL to ensure a proper match between the feeding rate and the juvenile shell's growth. In the later stages of juvenile growth, as the juvenile shell's water filtration capacity increased, the feeding rate was gradually increased to a maximum of 8 × 10⁴ cells / mL to meet the energy requirements for rapid mineralization of the shell and growth of the soft tissue.

[0035] In some respects, step 6) pre-sea acclimatization includes acclimatizing the indoor water temperature, salinity, and light exposure; In some embodiments, the acclimatization includes: slowly cooling the water at a rate of 1.0°C per day to gradually reduce the water temperature from the cultivation temperature to the same temperature in the sea area; controlling the salinity difference between the nursery pond and the target sea area to be ≤2‰; gradually increasing the light intensity to simulate natural diffused light of 500-1500 Lux; more preferably, during the acclimatization period, adding concentrated algal liquid rich in highly unsaturated fatty acids, vitamin C, and immunopolysaccharides to the feed.

[0036] In some preferred embodiments, step 6) pre-sea domestication also includes temporary rearing at sea: when the average shell height of the juvenile oysters reaches 500-800μm or more, the gill filaments are fully developed under a microscope, the foot movements are strong, the population mortality rate is less than 1%, and there are few miscellaneous algae on the substrate, temporary rearing at sea is carried out; the temporary rearing at sea is as follows: after the seedlings arrive at the sea area, the seedling bags are first suspended in the middle layer of the inner bay or floating raft with small waves and stable currents at a water depth of 3-5 meters for 3-7 days. After the juvenile oysters have fully adapted to the sea environment, they are then transferred to standard breeding cages for normal growth management.

[0037] The embodiments of this application are described in detail below with reference to examples. This application includes, but is not limited to, the following implementation methods. Any changes made to this application based on the prior art that do not depart from the essence of this application are still within the protection scope of this application.

[0038] This application establishes an indoor low-temperature circulating system in the north: the indoor low-temperature circulating water workshop has a water volume of approximately 200 cubic meters, the exterior walls are insulated with new fireproof and heat-insulating materials, and it is equipped with three to five sets of 20-horsepower refrigeration systems, an independent emergency power generation system, a cold water energy recovery system, a water treatment system, and a remote electronic monitoring system for temperature, water quality, video, etc.

[0039] Phase 1: Parent shellfish selection and low-temperature dormancy (mid-November) 1) Broodstock selection: Select healthy broodstock individuals with a shell height >10cm, aged 2-3 years, with regular shape, no damage, and robust development. After cleaning the shell surface to remove any attached substances, place them in a temporary holding tank at a density of approximately 20-30 individuals / m². 3 2) Low-temperature dormancy: Control the water temperature at 3-4℃ for 3-5 days; maintain a dark environment (<100 Lux) to simulate the environment under ice in deep winter. 3) Feeding: Feed a small amount of diatoms (Phaeodactylum tricornutum) according to the water quality monitoring results and the excretion of the parent shellfish, and siphon off the bottom and remove the waste daily.

[0040] Phase Two: Gradual Temperature Increase for Ripening (Late November - Late December) The temperature was slowly increased at a rate of 0.2-0.5℃ / day. When the temperature reached 5℃, 6℃, and 7℃, it was stabilized for 3-5 days. When the water temperature reached 8℃, a significant increase in food intake was observed. The temperature was then kept constant to promote maturation and synchronization.

[0041] During the maturation period, the light intensity was adjusted to 14L:10D (i.e., 14 hours of light: 10 hours of darkness, simulating the spring environment), and the light intensity was controlled at 500-800 Lux. A strategy combining natural bait and substitute bait was adopted. The natural bait included Algae Triangularis, Algae Rhizoctoniae, Algae Chrysophagus, and Algae Platyphyllum. The substitute bait included Vitamin E, emulsified fish oil, and marine red yeast.

[0042] The third stage, off-season induction of spawning and fertilization / hatching (early January) Maturity assessment: In early January, when the population gonadal index (GSI) > 15%, the female gonads are deep orange-red, and the male gonads are milky white, ovulation can be induced. The induction steps include: A combined induction method of "air drying + heat shock" was used. The parent scallops were air-dried for 1-2 hours (8-10℃) and then placed in warmed seawater 3-4℃ higher than the initial holding water temperature (i.e., 11℃-12℃). Fertilization and hatching were then carried out. When the number of eggs reached 40-50 per mL, the parent scallops were removed, and microscopic examination was performed to ensure that each egg was surrounded by 3-5 sperm cells. The mixed sperm were then evenly sprinkled, and the scallops were stirred every half hour.

[0043] The fourth stage, larval rearing and metamorphosis (early January to early February) 1) Larval Selection: Utilizing the floating habit of D-type scallop larvae, siphon selection is performed to remove dead eggs and deformed individuals from the bottom layer. Density Management: After selection, the D-type larvae are quantitatively adjusted to 10-15 larvae / mL per tank. From the late shell apex stage to the eyepoint larval stage, the density is gradually diluted to 2-3 larvae / mL to avoid density stress. Temperature and Water Quality Control: A gradient temperature increase is adopted, with the water temperature slowly raised from 12℃ at hatching at a rate of 0.5℃ / 3 days, eventually maintaining at 15℃ to promote metabolism and inhibit disease; water quality is maintained at a salinity of 30-32‰ and a pH of 8.0-8.2; the water exchange rate increases from 20% to 50% with age. 2) Graded Nutritional Enhancement: A "golden algae base, diatom growth promotion" strategy is implemented: In the early stage of larval cultivation, golden algae (…) are the main feed (…). Isochrysis zhanjiangensis ), ensuring the palatability of the initial feed; in the later stages of larval rearing, supplement with small unicellular algae such as diatoms (large crescent-shaped rhomboid algae or small crescent-shaped rhomboid algae) and flat algae. 3) Seedling collection and metamorphosis induction: when 20-30% of the larvae in the population show eye spots and their legs are active, enter the seedling collection period; place attachment substrate pretreated with biofilm (polyethylene mesh attachment substrate pre-soaked with photosynthetic bacteria for 24 hours), and at the same time adjust the light to weak diffused light (<100 Lux), using tactile attraction and photophobia to induce larval attachment and metamorphosis.

[0044] Phase 5: Juvenile Oyster Cultivation (Early February - Mid-March) 1) During the larval attachment period, initially after the attachment substrate is introduced, reduce the aeration rate (slight aeration) to minimize water flow disturbance and promote stable attachment of the larvae to the substrate. 2) During the juvenile growth period, once microscopic examination confirms complete larval attachment and metamorphosis into juveniles, significantly increase the aeration rate to ensure high dissolved oxygen levels in the water and promote even distribution of feed. Water quality control: As the juveniles' metabolism increases, gradually increase the daily water exchange rate, maintaining it at over 2 / 3 to effectively remove metabolic waste and prevent ammonia nitrogen accumulation. Nutritional fortification: Feed a diet supplemented with diatoms (large crescent-shaped algae, small crescent-shaped algae), flat algae, and other small unicellular algae; adjust the feeding amount according to the dynamic gradient of the juveniles' growth stages. During the early stages of juvenile oyster growth, the feeding amount should be controlled at a maximum of 4×10⁻⁶. 4 Cells / mL, ensuring feeding matching for newly metamorphosed juvenile clams. In the later stages of juvenile growth, as their water filtration capacity increases, the feeding amount is gradually increased to a maximum of 8 × 10⁶ cells / mL. 4 Cells / mL, to meet the energy requirements for rapid mineralization of juvenile shells and growth of soft tissue.

[0045] Phase 6: Pre-sea acclimatization (end of March) By gradually aligning indoor water temperature, salinity, and light levels with those of the sea area, stress and mortality rates during sea entry can be reduced. This includes the following aspects of acclimatization.

[0046] 1) Temperature Acclimation: Gradually reduce the water temperature from the cultivation temperature (approximately 15°C) to the ambient sea temperature (3°C-5°C) at a rate of 1.0°C per day. 2) Salinity Acclimation: Control the salinity difference between the nursery pond and the target sea area to ≤2‰; ideally ≤1‰. 3) Light Acclimation: Gradually increase the light intensity to simulate natural diffused light (500-1500 Lux). 4) Nutritional Fortification: During the acclimation period (7-10 days before release), add concentrated algae cake rich in highly unsaturated fatty acids, along with a formulated feed supplemented with vitamin C and immunomodulatory polysaccharides (glucan).

[0047] Then, the goods were removed from the warehouse and temporarily held at sea. Outgoing criteria: When the average shell height of juvenile oysters reaches 500-800μm or more, under a microscope, the gill filaments are fully developed, the foot movements are strong, the mortality rate of the population is less than 1%, and there are few miscellaneous algae on the substrate.

[0048] Temporary rearing at sea: After the seedlings arrive at the sea area, the seedling bags are first suspended in the inner bay or the middle layer of the raft (3-5 meters deep) where the wind and waves are small and the current is calm. They are temporarily reared for 3-7 days until the juveniles have fully adapted to the sea environment. Then, they are transferred to standard culture cages for normal growth and management.

[0049] Based on the aforementioned process, in the second half of 2024, this application constructed a 200-cubic-meter indoor seawater low-temperature circulation system in the sea area of ​​Changhai County, Dalian, and began collecting scallops for pilot-scale breeding experiments in November. The breeding experimental group was divided into 6 experimental groups and 1 control group in the seawater area. All experimental groups were based on the process of this invention. For detailed process differences, please refer to Table 1.

[0050] Table 1. Process settings for each experimental group .

[0051] Meanwhile, the applicant also included the traditional spring seedling process as a control group, unifying all experimental groups and the control group in terms of maturation time, gonad index (GSI), fertilization rate (%), hatching rate (%), sea release time, average shell height (μm) at the end of March, average shell height (cm) at the end of June, and summer survival rate (end of August), and comparing the results; in particular, after the second stage of maturation, scallop samples from each experimental group were randomly taken, the gonads were dissected and separated, and the gonadal mass and soft body mass were weighed, and the gonad index was calculated; after spawning and sperm release, gametes from each experimental group were artificially inseminated, samples were taken for observation and the number of eggs with the first polar body was counted, and the fertilization rate was calculated; after the fertilized eggs were further cultured to the D-shaped larval stage, the number of D-shaped larvae was counted, and the hatching rate was calculated. Formulas: Gonadal index (%) = Gonadal mass / Soft body mass × 100%; Fertilization rate (%) = Number of eggs with first polar body / Total number of observed eggs × 100%; Hatching rate (%) = Number of D-shaped larvae / Number of fertilized eggs × 100%. Specific test results and comparisons are shown in Table 2.

[0052] Table 2. Comparison of statistical results among groups .

[0053] The above results show that the off-season seedling cultivation method of this application can produce scallops with fast growth and high summer survival rate. As shown in Table 2, the off-season seedling cultivation method established in this patent application can advance the seedling production period and improve seedling size and stress resistance compared with traditional natural cultivation methods.

[0054] Furthermore, groups 2-6 further demonstrate the importance of each key step in this patent application. In group 2, without low-temperature dormancy, the maturation time of the broodstock was prolonged and the gonad index decreased, indicating that low-temperature dormancy is beneficial for unifying the physiological state of the broodstock. In group 3, the broodstock cultivation conditions of temperature, light, and feed were changed, resulting in a decrease in indicators such as gonad index, fertilization rate, hatching rate, and seedling size, indicating that gradient heating, light exposure, and compound nutrition enhancement are the core to improving the quality of maturation. Figure 1 and Figure 2The results were further verified by the sections of male and female gonadal tissues. It can be seen that the scallops cultivated by this process (Group 1) had full ovarian follicles, high oocyte maturity, more fully developed testes, and higher sperm density; while the gonadal maturity or tissue homogeneity of Groups 2 and 3 decreased to varying degrees. Group 4 did not induce spawning and sperm release in parent scallops through air drying, and the fertilization rate and hatching rate both decreased slightly, which affected the seedling size in the subsequent cultivation process, indicating that "air drying + heat shock" helps to improve the synchronization of spawning and sperm release; Group 5 did not involve dynamic feeding, and the shell height and summer survival rate decreased significantly in the later stage; Group 6 did not involve domestication before entering the sea, and the summer survival rate decreased significantly, indicating that the nutritional management of juvenile scallops and the process of acclimatization to the sea are crucial to the subsequent growth and survival of seedlings; however, the processes of Groups 5 and 6 had little impact on gonadal development (see Figure 1-2 Groups E and F in the text). Therefore, in Figure 1 and Figure 2 In the sections of male and female gonadal tissue, there was no significant difference in gonadal maturity and tissue homogeneity between group 1 and groups 4, 5, and 6.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for cultivating off-season scallop seedlings, characterized in that, Includes the following steps: 1) Parent shell screening; 2) Gradual heating to promote ripening; 3) Off-season induction of spawning, fertilization, and hatching; 4) Larval rearing and metamorphosis; 5) Juvenile shellfish cultivation; 6) Domestication before entering the sea.

2. The cultivation method according to claim 1, characterized in that, Step 1) Selection of parent shellfish includes a low-temperature dormancy process: the culture water temperature of the parent shellfish is controlled at 3℃-4℃ and maintained for 3-5 days; a dark environment of <100 Lux is maintained to simulate the environment under ice in deep winter. Preferably, step 1) of screening parent shellfish specifically includes the following steps: 1) Select healthy individuals with a shell height >10 cm, aged 2-3 years, with regular shape, no damage, and robust development; after cleaning the shell surface, temporarily raise them in a pool, controlling the density at 20-30 individuals / m². 3 ; 2) Maintain the water temperature at 3℃-4℃ for 3-5 days; keep the environment in complete darkness (<100 Lux) to simulate the environment under ice in deep winter; 3) Based on the water quality monitoring results and the excretion of the parent shellfish, feed them a small amount of diatoms and remove the bottom waste daily.

3. The cultivation method according to claim 1, characterized in that, Step 2) Gradual temperature increase for ripening includes the following steps: increasing the temperature at a rate of 0.2-0.5℃ / day, stabilizing the temperature for 3-5 days when the temperature reaches 5℃, 6℃, and 7℃ respectively, and when the water temperature reaches 8℃ and the feeding amount is significantly increased, then synchronizing the ripening process with constant temperature.

4. The cultivation method according to claim 3, characterized in that, In step 2) of gradient heating for ripening, the photoperiod is controlled at 14L:10D and the light intensity is controlled at 500-800 Lux. Preferably, during the ripening period, natural feed and substitute feed are provided. The natural feed includes *Phaeodactylum triangularis*, *Rhizophora spicata*, *Golden Algae*, and *Platycodon grandiflorus*, and the substitute feed includes vitamin E, emulsified fish oil, and marine red yeast.

5. The cultivation method according to claim 1, characterized in that, In step 3) off-season spawning and fertilization hatching, the off-season spawning is induced by a combination of "air drying + heat shock"; preferably, the combination induction is to air dry the parent scallops for 1-2 hours and then place them in warm seawater that is 3-4°C (i.e. 11°C-12°C) higher than the temporary rearing water temperature.

6. The cultivation method according to claim 5, characterized in that, Step 3) Off-season induction of spawning and fertilization hatching specifically includes the following steps: When the Group Gonadal Index (GSI) > 15%, the female gonads are deep orange-red and the male gonads are milky white, off-season induction is carried out; the parent shellfish are air-dried for about 1-2 hours and placed in water 3-4℃ higher than the temporary rearing water temperature (i.e., 11℃-12℃); when the spawning seawater reaches 40-50 eggs / mL, the parent shellfish are removed and examined under a microscope to control 3-5 sperm cells around each egg; the mixed sperm are evenly sprinkled and stirred once every half hour.

7. The cultivation method according to claim 1, characterized in that, Step 4) Larval cultivation and metamorphosis includes larval selection and seedling collection and metamorphosis induction; Preferably, the larval selection step includes: The siphon selection process utilizes the floating habit of D-type scallop larvae to remove dead eggs and deformed individuals from the bottom layer. After selection, the D-type larvae are quantitatively adjusted to a culture density of 10-15 larvae / mL per tank. From the late shell apex stage to the eyepoint larval stage, the density is gradually reduced to 2-3 larvae / mL to avoid density stress. Preferably, during larval selection, a gradient temperature increase is used, with the water temperature slowly raised from 12℃ at hatching time at a rate of 0.5℃ / 3 days, eventually maintaining at 15℃, promoting metabolism while inhibiting disease. Water quality is maintained at a salinity of 30-32‰ and a pH of 8.0-8.

2. The water exchange rate increases from 20% to 50% with age. More preferably, the larval rearing process employs a "golden algae as a base, diatoms for growth promotion" feeding strategy: in the early stages of larval rearing, golden algae are the primary feed to ensure palatability of the initial feed; in the later stages, diatoms and flat algae are added. The seedling collection and metamorphosis induction process includes: when 20-30% of the larvae in the population show eyespots and are active on their legs, the seedling collection period begins; an attachment substrate pretreated with biofilm is placed, and the light is adjusted to weak diffused light of <100 Lux, using tactile attraction and photophobia to induce larval attachment and metamorphosis.

8. The cultivation method according to claim 1, characterized in that, Step 5) juvenile shellfish cultivation includes: In the early stages after the larvae are placed on the attachment substrate, the aeration rate is reduced to achieve a micro-aeration state. After microscopic examination confirms that the larvae have completely attached and metamorphosed into juveniles, the aeration rate is increased. As the metabolism of the juveniles increases, the daily water exchange rate is gradually increased to maintain the water exchange rate at more than 2 / 3. Diatoms and flat algae are fed, and the feeding amount is adjusted according to the dynamic gradient of the juveniles' growth stages. Preferably, the dynamic gradient adjustment of the feeding amount is as follows: during the early stage of juvenile oyster growth, the feeding amount is controlled at a maximum of 4×10⁻⁶. 4 Cells / mL, ensuring feeding matching for newly metamorphosed juvenile clams; in the later stages of juvenile growth, as the clams' water filtration capacity increases, the feeding amount is gradually increased to a maximum of 8×10⁶ cells / mL. 4 Cells / mL, to meet the energy requirements for rapid mineralization of juvenile shells and growth of soft tissue.

9. The cultivation method according to claim 1, characterized in that, Step 6) Pre-sea acclimatization includes acclimatizing the indoor water temperature, salinity, and light exposure; Preferably, the acclimatization includes: slowly cooling the water at a rate of 1.0°C per day, gradually reducing the water temperature from the cultivation temperature to the same temperature in the sea area; controlling the salinity difference between the nursery pond and the target sea area to be ≤2‰; gradually increasing the light intensity to simulate natural diffused light of 500-1500 Lux; more preferably, during the acclimatization period, a concentrated algal solution rich in highly unsaturated fatty acids is added to the feed, along with vitamin C and immunopolysaccharides.

10. The cultivation method according to claim 1, characterized in that, Step 6) Pre-sea domestication also includes temporary rearing at sea: When the average shell height of the juvenile oysters reaches 500-800μm or more, the gill filaments are fully developed under a microscope, the foot movements are strong, the mortality rate of the population is less than 1%, and there are few miscellaneous algae on the substrate, temporary rearing at sea is carried out; the temporary rearing at sea is as follows: after the seedlings arrive at the sea area, the seedling bags are first suspended in the middle layer of the inner bay or floating raft with small waves and stable currents at a water depth of 3-5 meters for 3-7 days. After the juvenile oysters have fully adapted to the sea environment, they are then transferred to standard breeding cages for normal growth management.

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