Seedling raising method for improving crassostrea gigas gamete quality

Abstract

The invention belongs to the technical field of aquaculture, and particularly relates to a breeding method for improving the gamete quality of crassostrea gigas. The method comprises the following steps: selecting parent crassostrea gigas with non-developed gonads, carrying out lipopolysaccharide treatment on the parent crassostrea gigas, raising the temperature to promote maturity, and culturing filial generations by using gametes generated by the parents; the diameter of gamete eggs generated by the parent crassostrea gigas subjected to lipopolysaccharide treatment is increased, the total antioxidant capacity is improved, and the growth speed and the attachment metamorphosis capacity of generated offspring larvae are remarkably improved. The invention opens up a new way for solving the bottleneck problem of high death rate of larvae in the artificial breeding process of crassostrea gigas.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and in particular relates to a seedling cultivation method for improving the gamete quality of long oysters. Background Art

[0002] The long oyster is one of the most productive marine aquaculture shellfish in my country, with important economic and ecological value. In recent years, the frequent occurrence of long oyster diseases has caused huge economic losses, especially the high larval mortality rate has led to unstable seedling production, which has become one of the main bottlenecks that plague the high-quality development of the industry. Improving gamete quality and enhancing the disease and stress resistance of seedlings are important breakthroughs to ensure the healthy development of the oyster industry. Because long oysters lack an adaptive immune system and are mainly cultured in open / semi-open waters, it is difficult to control the occurrence of diseases through measures such as vaccination or water quality regulation. The development of immunopotentiators that can promote or induce immune defense responses and enhance the body's disease resistance has become an important direction for the prevention and control of aquatic shellfish diseases. Lipopolysaccharide has received widespread attention due to its significant potential in immune enhancement, immune memory formation, and drug delivery. At present, there are no reports on lipopolysaccharide improving the quality of long oyster gametes. Summary of the invention

[0003] The invention aims to provide a method for raising oysters for improving the gamete quality.

[0004] In order to achieve the above object, the specific technical solution adopted by the present invention is:

[0005] A method for raising oysters to improve the quality of gametes, wherein the method utilizes the immune enhancement principle to treat oyster broodstock with undeveloped gonads with lipopolysaccharides (LPS), and then raises them to maturity. The lipopolysaccharide treatment can improve the quality of their gametes and the rate of attachment and metamorphosis of their offspring. The total antioxidant capacity of the gametes produced by the method is improved, and the growth rate and the rate of attachment and metamorphosis of the offspring larvae are significantly improved.

[0006] Preferably, the oyster broodstock whose gonads have not yet developed are treated with lipopolysaccharide twice to promote the maturation of gametes and progeny larvae.

[0007] The concentration of the lipopolysaccharide was 0.5 g / L, and 100 μL was added each time.

[0008] Specifically, when the gonads of the long oyster broodstock are in an undeveloped state, 100 μL of 0.5 g / L lipopolysaccharide is injected into the adductor muscle, and after 7 days of feeding in normal seawater, a second injection is performed. The long oyster broodstock are then placed in seawater for temporary feeding and temperature cultivation, and the total antioxidant capacity of the gametes produced is improved. After the gonads of the long oyster broodstock mature, the gonads are dissected to obtain sperm and eggs, and artificial insemination is performed to produce larvae. The resulting offspring are placed in filtered seawater for cultivation, and high-quality juveniles are formed through embryonic development, attachment and metamorphosis.

[0009] The second object of the present invention is to provide a method for breeding oyster seed.

[0010] To achieve the above object, the specific technical scheme adopted by the present invention is: a method for breeding long oyster seed, including a parent cultivation stage, during which long oyster broodstock with undeveloped gonads are treated with lipopolysaccharide.

[0011] Preferably, the lipopolysaccharide treatment is administered twice to oyster broodstock whose gonads have not yet developed.

[0012] The concentration of the lipopolysaccharide was 0.5 g / L, and 100 μL was added each time.

[0013] Specifically, when the oyster broodstock are in the state of undeveloped gonads, 100 μL of 0.5 g / L lipopolysaccharide is injected into the adductor muscle, and after being temporarily fed in seawater for 7 days, a second injection is performed.

[0014] Compared with the prior art, the present invention has the following characteristics:

[0015] The seedling raising method of the present invention is simple to operate, low in cost and safe. The oyster broodstock are stimulated twice with lipopolysaccharide to promote gamete development, improve gamete quality, and enhance the attachment and metamorphosis ability of offspring larvae. Compared with the oysters of the control group injected with sterile filtered seawater, the gamete egg diameter of the oyster broodstock produced by the present invention using lipopolysaccharide stimulation is increased, the total antioxidant capacity is improved, the larvae growth rate is accelerated, and the attachment and metamorphosis ability is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The diameter of the spawning cells of the long oyster parent stimulated twice with lipopolysaccharide in Example 1. The significant difference between different groups is indicated by *: p<0.05.

[0017] Figure 2 The total antioxidant capacity of the male and female gametes produced by the long oyster parent stimulated twice with lipopolysaccharide in Example 1. The significant differences between different groups are indicated by *: p<0.05, **: p<0.01.

[0018] Figure 3The growth status of offspring produced by the oyster parent stimulated twice with lipopolysaccharide in Example 1. (A: shell length; B: shell height).

[0019] Figure 4 The figure shows the change in the attachment rate of the offspring larvae produced by the long oyster parent stimulated twice with lipopolysaccharide in Example 1. The significant difference between different groups is indicated by *: p<0.05. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and beneficial technical effect of the present invention clearer, the present invention is specifically described by examples below, it should be understood that the examples are only used to further illustrate the present invention, but the content of the present invention is not limited to the content involved in the examples, and it cannot be understood as a limitation on the scope of protection of the present invention, and the skilled person in the field can make some non-essential improvements and adjustments according to the above content of the present invention. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0021] Example 1

[0022] (1) Immune stimulation of long oysters: Long oyster broodstock were collected from the aquaculture waters of Zhangzidao, Dalian. 80 long oysters with undeveloped gonads were selected as breeding parents. When the long oyster broodstock were in the state of undeveloped gonads, 40 of them were injected with 100 μL of sterile filtered seawater per adductor muscle, and the other 40 were injected with 100 μL of 0.5 g / L lipopolysaccharide per adductor muscle. Then the above 80 broodstock were temporarily cultured in filtered seawater at 19°C. After 7 days, they were again immunized again using the same method and dosage.

[0023] (2) Ripening and artificial breeding of long oysters: The immunized long oysters were heated and cultivated in filtered seawater for 34 days. After the gonads matured, the broodstock were selected for dissection to obtain sperm and eggs, which were filtered using 200-mesh and 500-mesh sieves, respectively, 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 a 500-mesh sieve and then placed in a 200-L incubator for incubation. After the larvae developed to D-shaped larvae, they were selected and the larval density was controlled at 20 / mL. Golden algae were used for feeding and the water was changed once a day.

[0024] (3) Measurement of the diameter of oyster eggs: The gonads of the parents in the LPS-injected group and the sterile filtered seawater-injected group were dissected, and the eggs of the parents in the different treatment groups were placed in 20°C seawater for 30 minutes to mature. The maturation of the eggs was observed under a microscope, and the egg diameter was measured. The results showed that the diameter of the eggs produced by the LPS-injected parents (average 63.55 μm) was larger than that of the eggs produced by the control group (average 60.36 μm). Figure 1 ).

[0025] (3) Total antioxidant capacity test of male and female gametes of long oysters: Capacitated sperm and matured oocytes were selected and the total antioxidant capacity (T-AOC) test was performed according to the instructions of Nanjing Jiancheng kit. Figure 2 As shown, the total antioxidant capacity of sperm in the experimental group was 0.43nmol / gprot, and the total antioxidant capacity of oocytes was 0.45nmol / gprot, both of which were significantly higher than 0.37nmol / gprot (sperm) and 0.36nmol / gprot (oocytes) of the control group.

[0026] (4) Statistics on the growth of offspring larvae of long oysters: When the long oyster larvae began to develop into the D-form stage, the long oyster larvae of the experimental group and the control group were sucked out with a dropper every 2 days, the larval development was observed under a microscope, and the shell length and height of the larvae were measured. The results showed that the shell length of the larvae in the experimental group was significantly higher than that of the larvae in the control group starting from the third day after fertilization ( Figure 3 A), on the 5th day after fertilization, the shell height of the larvae in the experimental group began to be significantly higher than that in the control group ( Figure 3 B).

[0027] (5) Statistics of the attachment rate of offspring of long oysters: Scallop shells were used as the attachment base for larvae. Each group of 50 sterilized shells was hung in a string in a 50L culture barrel. When the proportion of eye-spot larvae in the lipopolysaccharide parent treatment group (experimental group) and the sterile seawater parent treatment group (control group) was 60%, the larvae were distributed in each culture barrel at a concentration of 0.3 / mL for 7 days. A mixed diet of Isochrysis algae, Platymonas, Chlorella, etc. was fed 3 times a day to ensure that the larvae obtained adequate nutrition. In addition, the water was changed once every 3 days to maintain the cleanliness and stability of the water quality. After 7 days, the attachment base was removed and the number of attached larvae was counted. The attachment rate is the percentage of the average number of attached larvae to the initial total number of larvae. Figure 4 As shown, the larval attachment rate of the lipopolysaccharide parent treatment group was 46.8%, and the larval attachment rate of the control group was 37.98%.

[0028] Therefore, through the method for treating the long oyster parent of the present invention, the diameter of the long oyster gamete eggs increases, the total antioxidant capacity is improved, and the quality of the long oyster gametes is improved. On this basis, the growth and development of the long oyster offspring larvae are significantly accelerated, and the attachment and metamorphosis ability is significantly enhanced.

[0029] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for raising oysters to improve the quality of gametes, comprising raising oyster broodstock, characterized in that: In the above-mentioned oyster broodstock culture, oyster broodstock with undeveloped gonads are treated with lipopolysaccharide and then cultured to maturity.

2. The method for raising oysters for improving the gamete quality according to claim 1, characterized in that: The oyster broodstock whose gonads had not yet developed were treated with lipopolysaccharide twice.

3. The method for raising oysters for improving the gamete quality according to claim 2, characterized in that: The lipopolysaccharide concentration is 0.5 g / L, 100 μL each time.

4. The method for raising oysters for improving the gamete quality according to claim 3, characterized in that: When the oyster broodstock was in a state of undeveloped gonads, 100 μL of 0.5 g / L lipopolysaccharide was injected into the adductor muscle, and after feeding in filtered seawater for 7 days, a second injection was performed.

5. A method for breeding oyster seed, comprising a parent cultivation stage, characterized in that: In the broodstock rearing stage, oyster broodstock whose gonads have not developed are treated with lipopolysaccharide.

6. The method for breeding oyster seedlings according to claim 5, characterized in that: The oyster broodstock whose gonads had not yet developed were treated with lipopolysaccharide twice.

7. The method for breeding oyster seedlings according to claim 6, characterized in that: The concentration of the lipopolysaccharide was 0.5 g / L, and 100 μL was added each time.

8. The method for breeding oyster seedlings according to claim 6, characterized in that: When the oyster broodstock was in a state of undeveloped gonads, 100 μL of 0.5 g / L lipopolysaccharide was injected into the adductor muscle, and after feeding in filtered seawater for 7 days, a second injection was performed.

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