A method for screening sea urchin gametes
Patent Information
- Application Number
- CN202611289610.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-25
- Publication Date
- 2026-10-30
AI Technical Summary
而海胆性腺品质，包括性腺是否残缺、畸形、颜色品相这些影响精卵质量和后代品质的实质性性腺性状指标，由于从外观难以分辨，所以在生产中一直不能利用
1.本发明创造性地建立了“初筛+解剖复检”的双重筛选流程，确保海胆种胆排放的精子、卵子品质与发育潜力，从而确保后代海胆的品质。
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Figure CN122997071A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of marine aquaculture seedling technology, and in particular to a method for screening sea urchin seed gallbladders. Background Technology
[0002] sea urchin( Echinoidea ) belongs to the phylum Echinodermata ( Echinodermata ), sea urchins ( Echinoidea Sea urchins are a typical type of marine benthic invertebrate, mainly distributed in shallow rocky reefs or algae-rich areas in temperate to subtropical seas. They are recognized as a precious marine delicacy due to their rich nutritional value and high medicinal and health benefits. In recent years, the market demand for sea urchins has expanded dramatically, leading to a significant increase in prices and rapid development of the sea urchin industry. The gonads of the sea urchin are its most important economic part, commonly known as "sea urchin roe." These gonads are rich in protein, unsaturated fatty acids, amino acids, and various vitamins, possessing high nutritional and commercial value, and have gradually formed a stable consumer market in my country. Due to their high price, sea urchin farming occupies an important position in the fisheries economy.
[0003] Studies have shown that growth traits and gonadal quality traits in sea urchins have moderate to high heritability, and there is a strong positive genetic correlation between them. Therefore, screening for growth traits and gonadal quality traits in sea urchin seedlings is essential for sea urchin seedling production. However, in current sea urchin seedling production, seedlings are primarily screened based on growth traits, including size, presence of damage, disease spots, and spine collapse. Since gonads are not visible in living sea urchins, selection is only based on observing whether the seedling releases reproductive cells, the continuity and amount of release, and whether the released egg cells clump together, to determine the suitability of gonadal development for fertilization. Substantial gonadal quality indicators, including whether the gonads are incomplete, deformed, and their color and appearance—which affect sperm, egg, and offspring quality—are difficult to discern from appearance and therefore cannot be utilized in production.
[0004] Therefore, in sea urchin seed production, there is an urgent need for a sea urchin seed selection method that can determine the quality of gonads in order to improve the quality of sea urchin reproductive cells, thereby improving the quality and survival rate of sea urchin seedlings. Summary of the Invention
[0005] To address the aforementioned limitations of existing technologies, the present invention aims to provide a method for screening sea urchin broodstock based on gonad quality. Specifically, sperm and eggs released from sea urchin broodstock are collected separately, and the source of the broodstock corresponding to each sperm and egg is recorded. First, the broodstock are preliminarily screened based on maturity by observing the continuity of sperm release, whether the released eggs clump together, and the amount of eggs released. This quickly eliminates individuals with overripe or underripe gonads, resulting in inferior reproductive cells, ensuring that the reproductive cells entering the fertilization stage have high viability. Then, the broodstock that pass the initial screening are dissected one by one, and a second screening is conducted by observing the morphology and color of the gonads. Broodstock with deformed, incomplete, or grayish-white gonads are removed. This effectively improves the quality of reproductive cells in sea urchin broodstock used for seedling cultivation, increases the metamorphosis survival rate of larvae at each stage, and thus improves seedling cultivation efficiency. This invention creatively establishes a dual screening process of "initial screening + dissection and re-examination," ensuring the quality and developmental potential of the sperm and eggs released from sea urchin broodstock, thereby ensuring the quality of offspring sea urchins.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for screening sea urchin seed bile, comprising the following steps: (1) Collect sea urchins without injury, disease spots, or spines that are not fallen as seed urchins. After temporarily culturing the seed urchins, induce ovulation or sperm release using the oxytocin stimulation method. Collect sperm or eggs from each seed urchin and record the source of the seed urchin corresponding to the sperm or eggs. (2) During the process of inducing ovulation or sperm release in male seedlings, preliminary screening of seedlings is carried out; specifically: during the process of inducing sperm release in male seedlings, the duration of sperm release is observed; during the process of inducing ovulation in female seedlings, the release of eggs is observed to see whether the released eggs are clumped together and the amount of eggs released. (3) Dissect the qualified seed gallbladders in the initial screening, and conduct a second screening of the seed gallbladders by observing the morphology and color of the gonads; (4) Select the sperm and eggs corresponding to the seed bile after the second screening for artificial insemination.
[0007] As a preferred option, in step (1), the diameter of the seed gallbladder is ≥5cm.
[0008] Preferably, in step (1), during the temporary rearing period of the seedlings, the density is controlled at 80-100 seedlings / m². 3 Light intensity ≤100 Lux, all seawater is changed once a day, and the food is one or more of the following: fresh kelp, wakame seaweed, salted kelp, and mussel meat.
[0009] As a preferred option, in step (1), the specific steps of the labor-inducing stimulation method are as follows: inject KCl solution into the periosteal membrane of the seed sac to stimulate the seed sac to release sperm or ovulate.
[0010] Furthermore, the KCl concentration is 0.4 mol / L-0.6 mol / L, and the injection volume for each seed is 2.5-3.5 ml.
[0011] As a preferred option, in step (2), if the duration of sperm release by the male sea urchin is ≥5min, the male sea urchin is considered to be qualified in the preliminary screening; otherwise, it is not qualified. If the eggs released by the female sea urchin are dispersed without clumping and the amount of eggs released at the end of ovulation is ≥10mL, the female sea urchin is considered to be qualified in the preliminary screening; otherwise, it is not qualified.
[0012] As a preferred option, in step (3), if the gonads of the seed gallbladder are regular in shape, without defects or deformities, and the color of the gonads is not grayish-white, then the second screening is qualified; otherwise, it is unqualified.
[0013] The beneficial effects of this invention are: 1. This invention creatively establishes a dual screening process of "initial screening + dissection and re-examination" to ensure the quality and developmental potential of sperm and eggs released by sea urchin seed urchins, thereby ensuring the quality of offspring sea urchins.
[0014] Specifically, sperm and eggs released from sea urchin broodstock are collected separately, and the source of the broodstock corresponding to the sperm and eggs is recorded. First, the broodstock are initially screened based on maturity by observing the continuity of sperm release, whether the released eggs clump together, and the amount of eggs released. This quickly eliminates individuals with overripe or underripe gonads that produce inferior reproductive cells, ensuring that the reproductive cells entering the fertilization process have high viability. Then, the broodstock that pass the initial screening are dissected one by one, and a second screening is conducted by observing the morphology and color of the gonads. The quality of sea urchin gonads has a medium to high heritability. Broodstock with gonadal malformations and grayish-white color have poor reproductive cell quality and will pass these gonadal characteristics on to offspring sea urchins. Furthermore, poor gonadal morphology and color will also affect the commercial value of sea urchins. This invention uses a two-stage screening process to remove malformed, incomplete, and grayish-white sea urchin seed urchins, which can effectively improve the quality of reproductive cells of seed sea urchins used for seedling cultivation, increase the floating rate of larvae after fertilization, reduce the loss of larvae at each developmental stage, increase the yield of subsequent sea urchin seedlings, and thus improve seedling cultivation efficiency.
[0015] Furthermore, a single female sea urchin can release millions of reproductive cells, and males release even more. If the reproductive cells of just one sea urchin with gonadal malformation or poor gonadal color are used in production, a large number of low-quality sea urchin seedlings will be generated. Therefore, the secondary screening process for seed urchins added in this invention is of great significance for improving the overall quality of the offspring sea urchin population.
[0016] 2. The method provided by this invention is simple, highly repeatable, and operable in production. This invention adds a dissection and rescreening step to the traditional initial screening of sea urchin seedlings without significantly extending the operation time, making it acceptable for production operations. Furthermore, this invention does not rely on complex experimental equipment or precision instruments and can be used directly in the production process; ordinary technicians can operate it independently after short-term training. In addition, this invention is applicable to all sea urchins, providing reliable technical support for the breeding of superior sea urchin varieties and the large-scale production of seedlings. Attached Figure Description
[0017] Figure 1 Preliminary screening images of sea urchin broodstock from the sea urchin *Echinochloa glabra*.
[0018] Figure 2 Images of sea urchins with incomplete or deformed gonads, such as *Echinochloa glabripennis*, during the secondary screening process. Detailed Implementation
[0019] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.
[0020] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0021] The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels.
[0022] In the embodiments and comparative examples of this invention, the urchin broodstock used were sea urchin broodstock temporarily held and preserved by Dalian Haibao Fishery Co., Ltd., and the shell diameter of each urchin was greater than 7 cm.
[0023] The duration of sperm release by a male sperm donor refers to the time during which the sperm donor continues to release sperm in the new germ cell collection cup after the sperm donor is observed to begin releasing sperm and is transferred to a new germ cell collection cup while sand-filtered seawater is continuously poured onto the sperm donor's mouthparts.
[0024] Example 1: (1) Collect 450 sea urchins with no damage, no disease spots, no fallen spines, and a shell diameter of more than 7 cm as seed urchins, and place them in a container with 90 urchins / m². 3 They were temporarily held at a density of 90 Lux and a light intensity of 90 Lux. During the holding period, the entire seawater was replaced once a day, and the food consisted of salted kelp and mussel meat. On September 30, 2025, five seed pods were randomly selected and dissected to measure the gonadal index in order to determine whether the seed pods were mature enough to be used for subsequent labor induction. The test results showed that the gonadal index of the selected seed pods was above 20%, indicating that the seed pods were mature enough to be used for subsequent labor induction. Two hundred sea urchin larvae without injury, disease spots, or fallen spines were randomly selected. The potassium chloride oxytocin stimulation method was used to induce ovulation or sperm release in the 200 larvae. The specific steps were as follows: 0.5 mol / L KCl solution was injected into the perimeter membrane of the larvae using a syringe, with an injection volume of 3 mL per larvae. Then, the larvae were placed with their reproductive openings facing down on a germ cell collection cup filled with sand-filtered seawater. Every 5 minutes, the larvae were moistened with filtered fresh seawater to keep them moist and allow them to release germ cells. (2) During the process of inducing ovulation or sperm release, the seed embryos are initially screened, such as... Figure 1 As shown, the details are as follows: During the process of inducing sperm ejaculation in male breeding stock, the duration of sperm ejaculation is observed to conduct preliminary screening. Specifically, when the breeding stock begins to ejaculate milky white semen, it is identified as a male breeding stock. It is then quickly transferred to a new germ cell collection cup, and sand-filtered seawater is poured onto its mouthparts. If the male breeding stock continues to ejaculate sperm for more than 5 minutes, it is considered to have passed the preliminary screening.
[0025] During the induction of ovulation in female germ cells, preliminary screening of female germ cells is conducted by observing whether the released eggs clump together and the amount of eggs released. Specifically, when the germ cell begins to release eggs, it is identified as a female germ cell; if the released eggs are observed to be dispersed without clumping, and the amount of eggs collected at the bottom of the germ cell collection cup at the end of ovulation is not less than 10 mL, then the female germ cell is considered to have passed the preliminary screening. After initial screening and testing, 11 female germ cells were not released; 94 female germ cells were ovulated; 15 female germ cells were ovulated with an amount of less than 10 mL or ovulated in clumps; 79 female germ cells met the initial screening requirements; 95 male germ cells released sperm; 76 male germ cells released sperm for a duration of ≥5 min; the total number of germ cells that passed the initial screening was 155. (3) After the initial screening, the female and male breeding stock were dissected and the morphology and color of the gonads were observed for secondary screening. If the gonads were regular in shape, without defects or deformities, and the color was not grayish-white, the secondary screening was considered successful. However, if the gonads were defective or deformed, such as… Figure 2 As shown; All 79 female breeding gallbladders were dissected, and 12 female gallbladders with malformed gonads, defects, or grayish-white color were removed. All 76 male breeding gallbladders were dissected, and 10 male gallbladders with malformed gonads, defects, or grayish-white color were removed. Therefore, after the second screening, the number of male breeding gallbladders was 66 and the number of female breeding gallbladders was 67, for a total of 133. (4) The remaining 67 female gallbladders with orange-yellow gonads and regular gonad morphology were mixed and transferred to a 110L large white barrel, and 160 million eggs were quantitatively counted. The sperm released by the 66 male gallbladders that passed the second screening were mixed and transferred to the above-mentioned large white barrel for artificial insemination of the eggs.
[0026] Comparative Example 1: The difference between this comparative example and Example 1 is that only the sea urchin oothecae are initially screened, while the temporary holding treatment is the same as in Example 1. Details are as follows: On September 30, 2025, 200 sea urchins without injury, disease spots, or lodging on their spines were selected from the broodstock after temporary rearing in Example 1. Ovulation or sperm release was induced in the broodstock using a potassium chloride oxytocin stimulation method. The specific steps were as follows: 0.5 mol / L KCl solution was injected into the periosteal membrane of each broodstock using a syringe, with an injection volume of 3 mL. The broodstock was then placed on a reproductive cell collection cup filled with sand-filtered seawater. Every 5 minutes, the broodstock was moistened with filtered fresh seawater to allow it to release reproductive cells. Then, the broodstock were preliminarily screened according to the method described in Example 1. The preliminary screening results were as follows: 13 germ cells that did not release any reproductive cells; 94 female germ cells that released eggs; 14 female germ cells that released clumps of eggs or released less than 10 mL of eggs at the end of ovulation; and 80 female germ cells that met the requirements. 93 male germ cells that released sperm; and 77 male germ cells that released sperm for a duration of ≥5 min. The eggs from the initially selected qualified female broodstock were mixed and transferred to a 110L container, and 189 million eggs were counted. The sperm from the initially selected qualified male broodstock were mixed and transferred to the same container for artificial insemination of the eggs.
[0027] Experimental Example 1: (1) The eggs were fertilized with sperm from *Echinocactus glaucus* obtained in Example 1. The specific steps were as follows: Collect the mixed semen and add it to a 110L white container containing 160 million eggs. Stir well and take samples for observation. Each fertilized egg has 30-50 sperm cells around it, yielding 145 million fertilized eggs. Add sand-filtered seawater to the container until the volume reaches 66L, stir well, and distribute the fertilized eggs evenly into 33 20L hatching tanks. Add 18L of 22-23℃ sand-filtered seawater to each tank. After 18 hours, the larvae float to the surface. Quantitatively measure the number of floating larvae, obtaining 103 million biarm larvae, with an average floating rate of 71.0%. Take the floating larvae and transfer them to 10 20-ton nursery ponds for larval rearing. The larval rearing density is 0.52 larvae / mL. Change the water once a day, replacing half the water each time. Feed with Chaetoceros muelleri. Quantitatively measure the number of planktonic larvae at the 6-arm larval stage, 8-arm larval stage, and late 8-arm larval stage. After 19 days of cultivation, 54 million late-stage larvae with 8 arms were obtained and used for pond splashing, entering their subsequent benthic attachment life.
[0028] The floating rate (%) = (number of bicarpal larvae / number of fertilized eggs) × 100%.
[0029] (2) The eggs were fertilized with sperm from *Ulmus glabra* obtained in Comparative Example 1. The specific steps were as follows: The mixed semen was added to a 110L tank containing 189 million eggs, stirred thoroughly, and samples were taken for observation. Each fertilized egg was found to have 30-50 sperm cells surrounding it, resulting in 167 million fertilized eggs. Sand-filtered seawater was added to the tank to bring the volume to 66L, and the mixture was stirred. The fertilized eggs were then evenly distributed into 33 20L hatching tanks. Each tank was given 18L of 22-23℃ sand-filtered seawater. After 18 hours, the larvae floated to the surface. A quantitative count of the floating larvae was taken, yielding 101 million biarm larvae. These floating larvae were transferred to 10 20-ton nursery ponds for larval rearing. The larval rearing density was 0.51 larvae / mL. Half of the water was changed daily, and *Chaetoceros muelleri* was used as feed. The planktonic larvae were quantitatively measured at the 6-arm larval stage, the 8-arm larval stage, and the late 8-arm larval stage. After 19 days of cultivation, 40 million late-stage larvae with 8 arms were obtained and used for pond splashing, entering their subsequent benthic attachment life.
[0030] The number of planktonic larvae per unit volume (mL) was quantified for each rearing stage, i.e., the number of planktonic larvae / total volume of the rearing pond. The results are shown in Table 1.
[0031] Table 1. Quantitative results of sea urchin larvae at different developmental stages before splashing. When the larvae develop primordia and enter the late 8-arm stage, they are transferred to nursery ponds containing benthic diatom feed for benthic cultivation. Eight ponds with identical light and other cultivation conditions were selected from a row of 10-ton nursery ponds. Four ponds were used to cultivate *Echinocactus glaucus* larvae (Example 1), and the other four were used to cultivate *Echinocactus glaucus* larvae (Comparative Example 1). Each pond contained 7 tons of water and 90 benthic diatom feed baskets, each containing 14 feed plates. Larvae were scooped out with a scoop, gently scattered in the center of the pond, close to the water surface. The density was 0.2 larvae / mL. During the period before the larvae fully attached, the water was completely changed once daily using a 260-mesh filter. Approximately 10 days after stocking, once the larvae began to open, benthic diatom feed baskets were placed in the nursery ponds. When the benthic diatoms were exhausted, the larvae were anesthetized with a garlic-infused seawater solution for larval detachment. The detachment quantity is shown in Table 2.
[0032] Table 2. Quantitative results of sea urchin seedling stripping. As shown in Tables 1 and 2, the sea urchin seed selection method of the present invention can improve the cultivation effect of sea urchin larvae at all stages, reduce sea urchin larvae loss, and improve the sea urchin seedling cultivation effect. Compared with Comparative Example 1, the number of sea urchin seedlings obtained by peeling in Example 1 increased by 26%. The increase in the number of juvenile urchins directly improved the survival rate and breeding efficiency during the seedling stage.
[0033] This demonstrates that early sea urchin embryos are extremely sensitive to the quality of the parent gonads. If the parent gonads have tissue defects, functional decline, or poor quality, it often leads to a decrease in fertilization rate and an increase in early embryonic malformation rate, thus reducing the number of juvenile sea urchins bred. By observing the gonads after the release of reproductive cells from the parent sea urchin and removing malformed, defective, or poorly colored parents, these risks can be effectively avoided, significantly increasing the number of normally developing juvenile sea urchins.
[0034] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A method for screening sea urchin broodstock, characterized in that, Includes the following steps: (1) Collect sea urchins without injury, disease spots, or spines that are not fallen as seed urchins. After temporarily culturing the seed urchins, induce ovulation or sperm release using the oxytocin stimulation method. Collect sperm or eggs from each seed urchin and record the source of the seed urchin corresponding to the sperm or eggs. (2) During the process of inducing ovulation or sperm release in male seedlings, preliminary screening of seedlings is carried out; specifically: during the process of inducing sperm release in male seedlings, the duration of sperm release is observed; during the process of inducing ovulation in female seedlings, whether the eggs clump together is observed, and after ovulation, the amount of eggs released is observed; (3) Dissect the qualified seed gallbladders in the initial screening, and conduct a second screening of the seed gallbladders by observing the morphology and color of the gonads; (4) Select the sperm and eggs corresponding to the seed bile after the second screening for artificial insemination.
2. The sea urchin seed bile screening method as described in claim 1, characterized in that, In step (1), the shell diameter of the seed gallbladder is ≥5cm.
3. The sea urchin seed bile screening method as described in claim 1, characterized in that, In step (1), during the temporary rearing period of the seedlings, the density is controlled at 80-100 seedlings / m². 3 Light intensity ≤100 Lux, all seawater is changed once a day, and the food is one or more of the following: fresh kelp, wakame seaweed, salted kelp, and mussel meat.
4. The sea urchin seed bile screening method as described in claim 1, characterized in that, In step (1), the specific steps of the labor-inducing stimulation method are as follows: inject KCl solution into the periosteal membrane of the seed sac to stimulate the seed sac to release sperm or ovulate.
5. The sea urchin seed bile screening method as described in claim 4, characterized in that, The KCl concentration is 0.4 mol / L-0.6 mol / L, and the injection volume for each seed is 2.5-3.5 ml.
6. The sea urchin seed bile screening method as described in claim 1, characterized in that, In step (2), if the duration of sperm release by the male sea urchin is ≥5min, the male sea urchin is considered to be qualified in the preliminary screening; otherwise, it is not qualified. If the eggs released by the female sea urchin are dispersed without clumping and the amount of eggs released at the end of ovulation is ≥10mL, the female sea urchin is considered to be qualified in the preliminary screening; otherwise, it is not qualified.
7. The sea urchin seed bile screening method as described in claim 1, characterized in that, In step (3), if the gonads of the seed gallbladder are regular in shape, without defects or deformities, and the color of the gonads is not grayish-white, then the second screening is qualified; otherwise, it is unqualified.