Breeding method for improving quality of long oyster gametes
By injecting lipopolysaccharide into the gonads of Pacific oyster parent oysters before they develop, gamete development is promoted, which solves the problem of high mortality rate of Pacific oyster larvae, significantly improves gamete quality and offspring larval growth rate, and enhances the disease resistance of seedlings.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- DALIAN OCEAN UNIV
- Filing Date
- 2025-01-10
- Publication Date
- 2026-05-29
AI Technical Summary
The high mortality rate of oyster larvae leads to unstable seed production. Existing technologies are insufficient to effectively control the disease through vaccination or water quality regulation, and there is a lack of methods to enhance gamete quality.
When the gonads of the Pacific oyster parent oysters are not yet developed, they are treated with lipopolysaccharide by injecting 0.5 g/L of lipopolysaccharide solution into the adductor muscle to promote gamete development. The parent oysters are then temporarily raised in seawater, and the treatment is repeated twice to improve the quality of the gametes.
It improved the total antioxidant capacity of gametes, enhanced the growth rate and attachment metamorphosis rate of offspring larvae, and improved the disease resistance of seedlings.
Smart Images
Figure CN119699249B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of aquaculture technology, specifically relating to a method for improving the quality of gametes in Pacific oysters. Background Technology
[0002] The Pacific oyster is one of the most produced marine aquaculture shellfish in my country, possessing significant economic and ecological value. In recent years, frequent diseases affecting Pacific oysters have caused substantial economic losses, particularly the high mortality rate of larvae, leading to unstable seedling production and becoming a major bottleneck hindering the industry's high-quality development. Improving gamete quality and enhancing seedling disease resistance are crucial breakthroughs for ensuring the healthy development of the oyster industry. Because Pacific oysters lack an adaptive immune system and are primarily cultured in open / semi-open waters, disease control through vaccination or water quality regulation is difficult. Developing immune enhancers that can promote or induce immune defense responses and enhance disease resistance has become an important direction for disease control in aquatic shellfish. Lipopolysaccharides (LPS) have attracted widespread attention due to their significant potential in immune enhancement, immune memory formation, and drug delivery; however, there are currently no reports on LPS improving Pacific oyster gamete quality. Summary of the Invention
[0003] The purpose of this invention is to provide a method for raising seedlings that improves the quality of gametes in the Pacific oyster.
[0004] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:
[0005] A method for improving the quality of gametes in Pacific oysters involves treating gonadally-undeveloped Pacific oyster broodstock with lipopolysaccharides (LPS) during broodstock rearing, utilizing the principle of immune enhancement, and then cultivating them to maturity. LPS treatment improves gamete quality and offspring attachment metamorphosis rate. The gametes produced by this invention exhibit increased total antioxidant capacity, and the growth rate and attachment metamorphosis rate of offspring larvae are significantly enhanced.
[0006] Preferably, the parent oysters of the Pacific oyster whose gonads have not yet developed are given two lipopolysaccharide treatments to promote maturation and the production of gametes and offspring larvae.
[0007] The concentration of the lipopolysaccharide is 0.5 g / L, and the dosage is 100 μL each time.
[0008] Specifically, when the broodstock of the Pacific oyster is in an undeveloped gonadal state, 100 μL of lipopolysaccharide at a concentration of 0.5 g / L is injected into the adductor muscle. After being temporarily reared in normal seawater for 7 days, a second injection is performed. The broodstock are then temporarily reared in seawater with feed and heated, resulting in gametes with increased total antioxidant capacity. Once the gonads of the broodstock have matured, the gonads are dissected to obtain sperm and egg cells, which are then artificially inseminated to produce larvae. The offspring are then reared in filtered seawater, undergoing embryonic development and attachment metamorphosis to form high-quality juvenile oysters.
[0009] The second objective of this invention is to provide a method for breeding Pacific oyster seedlings.
[0010] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: a method for breeding Pacific oyster seedlings, including a parent stock cultivation stage, in which undeveloped Pacific oyster parent oysters are treated with lipopolysaccharide.
[0011] Preferably, the parent oysters of the Pacific oyster whose gonads have not yet developed are given two lipopolysaccharide treatments.
[0012] The concentration of the lipopolysaccharide is 0.5 g / L, and the dosage is 100 μL each time.
[0013] Specifically, when the parent oysters of the Pacific oyster are in an undeveloped gonadal state, 100 μL of lipopolysaccharide at a concentration of 0.5 g / L is injected into the adductor muscle. After being temporarily kept in seawater with feed for 7 days, a second injection is performed.
[0014] Compared with the prior art, the present invention has the following characteristics:
[0015] The seedling cultivation method of this invention is simple to operate, low in cost, and safe. It utilizes two rounds of lipopolysaccharide stimulation on the broodstock of *Crassostrea gigas* to promote gamete development, improve gamete quality, and enhance the attachment and metamorphosis abilities of offspring larvae. Compared to the control group of *Crassostrea gigas* injected with sterile filtered seawater, the gametes produced by the broodstock of this invention, stimulated by lipopolysaccharide, exhibit increased egg diameter, improved total antioxidant capacity, faster larval growth rate, and significantly enhanced attachment and metamorphosis abilities. Attached Figure Description
[0016] Figure 1 The change in the diameter of oocytes produced by the parent oyster *Crassostrea gigas* after two lipopolysaccharide stimulations in Example 1. Significant differences between groups are indicated by *: p < 0.05.
[0017] Figure 2 The total antioxidant capacity changes in male and female gametes produced by the parent oysters stimulated twice with lipopolysaccharide in Example 1. Significant differences between different groups are indicated by *: p < 0.05, **: p < 0.01.
[0018] Figure 3The growth status of offspring produced from the parent oysters of the Pacific oyster that underwent two rounds of lipopolysaccharide stimulation in Example 1. (A: shell length; B: shell height).
[0019] Figure 4 The attachment rate of offspring larvae from the parent oyster *Crassostrea gigas* after two lipopolysaccharide stimulations in Example 1 is shown. Significant differences between groups are indicated by *: p < 0.05. Detailed Implementation
[0020] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention is described in detail below through embodiments. It should be understood that these embodiments are only for further illustration of the invention, and the scope of the invention is not limited to the content described in the embodiments, nor should they be construed as a limitation on the scope of protection of the invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above description of the invention. Unless otherwise specified, all reagents and materials used in the following embodiments are commercially available.
[0021] Example 1
[0022] (1) Immunostimulation of Pacific oyster parent stock: Pacific oyster parent stock were collected from the aquaculture area of Zhangzidao Island, Dalian. Eighty Pacific oysters with undeveloped gonads were selected as breeding parent stock. When the Pacific oyster parent stock was in the undeveloped gonadal state, 40 of them were injected with 100 μL of sterile filtered seawater into the adductor muscle of each parent stock, and the other 40 were injected with 100 μL of lipopolysaccharide at a concentration of 0.5 g / L into the adductor muscle of each parent stock. Then, the above 80 parent stock were temporarily kept in filtered seawater at 19℃. After 7 days, a second immunization injection was performed using the same method and dosage.
[0023] (2) Maturation and artificial seedling cultivation of Pacific oyster parent stock: Immunized Pacific oyster parent stock was cultured in filtered seawater for 34 days under elevated temperature to promote maturation. After the gonads matured, the parent oysters were selected for dissection to collect sperm and eggs, which were then filtered through 200-mesh and 500-mesh silk screens, and then poured into filtered seawater for maturation. The sterile filtered seawater injection group (control group) and the lipopolysaccharide injection group (experimental group) were artificially inseminated at a ratio of 1:5 for sperm and eggs. Ten minutes after fertilization, the fertilized eggs were washed three times with 500-mesh silk screens and then placed in a 200L incubator for hatching. After the larvae developed into D-shaped larvae, the best larvae were selected, and the larval density was controlled at 20 larvae / mL. Golden algae were used for feeding, and the water was changed once a day.
[0024] (3) Measurement of Oyster Egg Diameter: The gonads of the parents in the lipopolysaccharide injection group and the sterile filtered seawater injection group were dissected. Oyster eggs from the different treatment groups were placed in seawater at 20℃ for 30 minutes to mature. The maturation of the oyster eggs was observed under a microscope, and the egg diameter was measured. The results showed that the egg diameter of the lipopolysaccharide-injected parents (average 63.55 μm) was larger than that of the control group parents (average 60.36 μm). Figure 1 ).
[0025] (3) Detection of total antioxidant capacity of male and female gametes of Pacific oyster: Capacitated sperm and matured oocytes were selected for total antioxidant capacity (T-AOC) detection according to the instructions of the Nanjing Jiancheng reagent kit. Figure 2 As shown, the total antioxidant capacity of sperm in the experimental group was 0.43 nmol / gprot, and the total antioxidant capacity of oocytes was 0.45 nmol / gprot, both of which were significantly higher than those of the control group (0.37 nmol / gprot for sperm and 0.36 nmol / gprot for oocytes).
[0026] (4) Statistics on the growth status of Pacific oyster offspring larvae: Starting from the D-stage of Pacific oyster larvae development, larvae from both the experimental and control groups were collected every two days using a dropper. Larval development was observed under a microscope, and shell length and height were measured. Results showed that the shell length of the experimental group larvae was significantly higher than that of the control group larvae starting on the third day after fertilization. Figure 3 A) On the 5th day after fertilization, the shell height of the experimental group larvae was significantly higher than that of the control group larvae. Figure 3 B).
[0027] (5) Attachment rate statistics of Pacific oyster offspring: Scallop shells were used as attachment substrates for larvae. Each group consisted of 50 sterilized shells, suspended in strings in 50L culture tanks. When the proportion of eye-stage larvae in the lipopolysaccharide parent treatment group (experimental group) and the sterile seawater parent treatment group (control group) reached 60%, larvae were distributed in each culture tank at a concentration of 0.3 larvae / mL for 7 days. *Isochrysis*, *Platycodon*, and *Chlorella* were fed three times daily to ensure sufficient nutrition for the larvae. In addition, the water was changed every three days to maintain water quality cleanliness and stability. After 7 days, the attachment substrates were removed, and the number of attached larvae was calculated. The attachment rate was the percentage of the average number of attached larvae to the initial total number of larvae. Figure 4 As shown, the larval attachment rate in the lipopolysaccharide parent treatment group was 46.8%, while the larval attachment rate in the control group was 37.98%.
[0028] Therefore, through the parent oyster treatment method of the present invention, the egg diameter of the oyster gametes increases, the total antioxidant capacity is improved, and the quality of the oyster gametes is enhanced. Based on this, the growth and development of the oyster offspring larvae are significantly accelerated, and their attachment and metamorphosis abilities are significantly enhanced.
[0029] The above-described embodiments are merely one implementation of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.
Claims
1. A method for improving the quality of gametes in the Pacific oyster, comprising the cultivation of Pacific oyster broodstock, characterized in that: In the cultivation of Pacific oyster broodstock, Pacific oyster broodstock whose gonads have not yet developed are given two lipopolysaccharide treatments, and then raised to promote maturation until maturity. The two lipopolysaccharide treatments are performed by injecting lipopolysaccharide into the adductor muscle of the Pacific oyster broodstock when the gonads are in an undeveloped state, and then temporarily raising them in filtered seawater for 7 days before the second injection.
2. The method for improving the quality of gametes in *Oryctosoma longicornis* according to claim 1, characterized in that: The concentration of the lipopolysaccharide is 0.5 g / L, and the dosage is 100 μL each time.
3. A method for breeding Pacific oyster seedlings, including a parent stock cultivation stage, characterized in that: During the parent stock cultivation stage, the oysters whose gonads have not yet developed are given two lipopolysaccharide treatments to improve the quality of the oyster gametes, and then the temperature is raised to promote maturation until maturity; The two lipopolysaccharide treatments involved injecting lipopolysaccharide into the adductor muscle of the oyster parent oysters while their gonads were still undeveloped, followed by a second injection after they were temporarily kept in filtered seawater and fed for 7 days.
4. The method for breeding Pacific oyster seedlings according to claim 3, characterized in that: The concentration of the lipopolysaccharide is 0.5 g / L, and the dose is 100 μL each time.