Crassostrea gigas fry breeding method capable of improving immunity of D-type larvae
By treating the early D-type larvae with 0.053 g/L ammonium chloride solution for 24 hours, the problem of high mortality of long oyster seedlings was solved, their immunity and quality were improved, and the mortality rate was reduced.
Patent Information
- Application Number
- CN202510887755.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-10-10
AI Technical Summary
During the cultivation of long oyster seed, the mortality rate and immunity of type D larvae are low, especially before the formation of shell-top larvae, the mortality rate can be as high as over 50%. Existing technology has failed to effectively utilize ammonium chloride to improve their immunity.
During the cultivation of long oyster seed, the early D-type larvae were treated with 0.053 g/L ammonium chloride solution for 24 hours, and then the larvae were transferred to water without ammonium chloride for continued cultivation, which significantly improved the autophagy level and immune gene expression of the D-type larvae.
It significantly reduces the mortality rate of D-type larvae, enhances their immune response ability and inflammation-related gene expression, improves the quality and immunity of long oyster seedlings, and is simple to operate and low in cost.
Smart Images
Figure CN120753214A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of shellfish farming, and in particular to a method for cultivating long oyster seed capable of improving the immunity of D-type larvae. Background Art
[0002] Long oyster ( Crassostrea gigas The oyster (Crassostrea gigas) is an important marine aquaculture shellfish with extremely high economic value. However, during the cultivation of long oyster seed, numerous adverse factors, such as marine pollution, climate disasters, predators, and pathogens, can cause large-scale mortality in larvae. Especially during the critical developmental stage of the D-type larvae, before the formation of the shell-top larvae, larval mortality rates rise sharply, sometimes exceeding 50% and, in extreme cases, even reaching over 80%.
[0003] Ammonium chloride (NH₄Cl) is a major byproduct of the soda ash production process. It is a white crystalline solid that readily absorbs moisture, agglomerates, decomposes easily upon heating, and is soluble in water, alcohol, and glycerol. It has been widely used in various technical fields. In agriculture, ammonium chloride is often used as a fertilizer. It not only helps crop growth and increases yield, but also effectively reduces the acidity of fruit while increasing its sugar and vitamin C (Vc) content, significantly enhancing its taste and nutritional value. In animal husbandry, ammonium chloride is used as a feed additive for ruminants, helping to alleviate heat stress in poultry exposed to high temperatures and preventing conditions such as hypocalcemia, thereby ensuring their healthy growth. In aquaculture, adding ammonium chloride to algae culture medium and using this algae as a feed additive significantly boosts the immunity of waterfowl, providing strong support for the sustainable development of aquaculture. Furthermore, ammonium chloride is used in medical technology as an expectorant.
[0004] Because the long-necked oyster is a mollusk, its digestive system differs significantly from that of ruminants or monogastrics. Its primary feeding method is to filter plankton and organic detritus from the water, relying on its gills and digestive glands for nutrient absorption. Its nitrogen requirements and utilization mechanisms in feed differ from those of terrestrial animals. Oysters, in particular, live in an aquatic environment, where water quality and pH are crucial for their survival and metabolism. Ammonium chloride, the nitrogen form of ammonium nitrogen, is easily converted into ammonia in water. Excessive ammonia is toxic to aquatic organisms and can harm the gills and respiratory system of long-necked oysters, even causing death. Therefore, to date, there have been no reports of treating D-type larvae with a certain concentration of ammonium chloride during seed cultivation to effectively enhance their immunity. Summary of the Invention
[0005] The present invention aims to solve the above-mentioned technical problems existing in the prior art and provides a method for cultivating long oyster seedlings which can improve the immunity of D-type larvae.
[0006] The technical solution of the present invention is: fertilized eggs of long oysters are obtained and cultured by an artificial breeding method; when the fertilized eggs of long oysters develop into the early stage of D-type larvae, they are treated with aquaculture water with a final ammonium chloride concentration of 0.053 g / L for 24 hours; and then the D-type larvae are moved to aquaculture water without ammonium chloride for continued cultivation.
[0007] Preferably, the treatment of the D-type larvae with a final ammonium chloride concentration of 0.053 g / L in aquaculture water for 24 hours comprises spraying an ammonium chloride solution into the aquaculture water of the early stage D-type larvae to achieve a final ammonium chloride concentration of 0.053 g / L, and aquaculture the D-type larvae for 24 hours.
[0008] The present invention is to treat the D-type larvae with a final concentration of ammonium chloride of 0.053 g / L during the cultivation of long oyster seedlings. The D-type larvae are treated with aquaculture water with a final concentration of ammonium chloride of 0.053 g / L for 24 hours, and then the D-type larvae are transferred to aquaculture water without ammonium chloride for continued cultivation. The present invention can significantly improve the autophagy level of D-type larvae, promote the proliferation of D-type larvae's blood lymphocytes, and enhance the D-type larvae's immune response to viruses. In addition, ammonium chloride can also induce the expression of inflammation-related genes in D-type larvae, thereby comprehensively improving the immunity of long oyster D-type larvae, effectively reducing larval mortality, and improving the quality of long oyster seedlings. At the same time, the present invention also has the advantages of simple operation and low cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 It is a statistical diagram of the mortality rate and deformity rate of D-type larvae in the embodiment of the present invention and the control group.
[0010] Figure 2 Graph showing changes in mRNA levels of immune-related genes in D-type larvae of the examples of the present invention and the control group.
[0011] Figure 3 Graph showing changes in the protein levels of the autophagy-related gene LC3 in D-type larvae of the Example of the present invention and the control group. DETAILED DESCRIPTION
[0012] The present invention provides a method for cultivating long oyster seedlings capable of improving the immunity of D-type larvae. The method comprises the following steps: artificially inseminating and culturing long oysters according to a conventional method in the prior art; when the fertilized eggs of the long oysters develop into D-type larvae, calculating the amount of ammonium chloride powder according to the volume of seawater for cultivating the long oyster larvae; preparing a high-concentration ammonium chloride solution by mixing ammonium chloride and seawater; and spraying the solution into the cultivation water of the early stage of the D-type larvae to obtain a final ammonium chloride concentration of 0.053 g / L; cultivating the D-type larvae for 24 hours, filtering the solution with a sieve silk, and transferring the D-type larvae to a cultivation water without ammonium chloride and continuing to cultivate the solution into seedlings according to a conventional method.
[0013] The ammonium chloride used in the embodiment of the present invention is produced by Beijing Solebow Technology Co., Ltd., with the product number A7320.
[0014] experiment: Fertilized oyster eggs (Crassostrea gigas) were obtained through artificial seedling cultivation and divided equally into two groups, each containing 50 L of water: a control group (Blank) and a group treated with NH₄Cl (NH₄Cl). The control group was cultured according to conventional methods, while the group treated with the methods described in the examples of the present invention was treated.
[0015] All experiments in the present invention were set up in triplicate, and the obtained data were expressed as mean ± SD. One-way analysis of variance and LSD multiple comparison were performed using IBM SPSS Statistics 20 software, and graphs were drawn using Origin Graph and GraphPad Prism 8. p Values less than 0.05 are considered to be significantly different; p The difference between the two groups of data was analyzed using Student's t test (" " indicates p < 0.05;" "express p < 0.01;" "express p < 0.001;" "express p < 0.0001).
[0016] Experiment 1: Detection of mortality and deformity rate of D-type oyster larvae in each experimental group After the D-type larvae were treated with ammonium chloride for 24 hours, samples of the D-type larvae from the control group and the group according to the present invention were collected for statistical analysis of the mortality and deformity rates of the larvae. The specific operation was as follows: 1 mL of seawater was drawn from 50 L of aquaculture water, filtered through a sieve to obtain a D-type larvae sample, and then 1 μL of Gru's reagent was added to the sample. The treated samples of each group were observed under a microscope and photographed, and the mortality and deformity rates of the seedlings were statistically analyzed. The results are as follows: Figure 1 shown. Figure 1 A: comparison of the mortality rate of D-type larvae in the control group and the group according to the present invention; B: comparison of the deformity rate of D-type larvae in the control group and the group according to the present invention; C: normal D-type larvae; D: deformed D-type larvae.
[0017] The results showed that after the D-type larvae were treated with ammonium chloride for 24 h, the mortality rate of the D-type larvae in the Example group of the present invention was significantly reduced, which was 0.21 times that of the control group ( p < 0.001) ( Figure 1A); Compared with the control group, there was no significant difference in the deformity rate in the embodiment group of the present invention ( Figure 1 B). Deformities of D-type oyster larvae include: convex shell, scalloped shell, incomplete shell, mantle abnormality, and arrested embryonic development. Figure 1 C is the normal D-type larva observed in this experiment. Figure 1 D is the D-type larva with mantle abnormality observed in this experiment.
[0018] Experiment 2: Effects of experimental groups on the expression of immune-related genes in D-type larvae of long oysters After the D-type larvae were treated with ammonium chloride for 24 hours, samples were collected from both the control and the inventive example groups for RNA extraction. The expression of immune and developmental genes in the D-type oyster larvae treated with ammonium chloride was then examined. RT-qPCR (reverse transcription quantitative polymerase chain reaction) was used to examine changes in mRNA expression of genes associated with immunity, such as hematopoiesis, antiviral therapy, and autophagy, as well as developmental genes.
[0019] The specific operations are as follows: After 24 hours of ammonium chloride treatment of D-type larvae from the present invention group, larval samples from each group were collected using a 300-mesh sieve into 50 mL tubes. The samples were centrifuged at 8000 rpm for 5 minutes, the seawater was aspirated, and the tubes were thoroughly resuspended in 20 mL of PBS. The tubes were centrifuged at 8000 rpm for 5 minutes, the PBS was aspirated, and this process was repeated three times before being aliquoted into 1.5 mL tubes. RNA samples were added with 500 μL of Trizol, snap-frozen in liquid nitrogen, and stored at -80°C.
[0020] qRT-PCR technology was used to detect the mRNA expression changes of genes related to autophagy, hematopoiesis, antiviral and inflammation, which have been proven to be related to immunity. -ΔΔCt The reaction system is shown in Table 1: Table 1 RT-qPCR reaction system Table 1 Reaction system for RT-qPCR
[0021] Note: The reaction program is 95℃, 30 s (1 cycle); 95℃, 5 s, 60℃, 34 s (40 cycles).
[0022] The gene expression results of the control group and the embodiment of the present invention group are as follows Figure 2 shown. Figure 2 Middle AE: autophagy-related genes CgBeclin1, Cg ATG5, Cg ATG16L, Cg LC3 and Cg P62; FK: hematopoietic related genes Cg Notch1, Cg TNF-2, Cg BMP-7, Cg CDC-6, Cg CDK-2 and Cg Runx; LN: antiviral-related genes Cg TRIM, Cg Mx1 and Cg IFI44;OQ: inflammation-related genes Cg IL17-1, Cg IL17-5 and Cg GSDME.
[0023] The experimental results showed that after 24 h of ammonium chloride treatment of long oyster D-type larvae, autophagy-related genes Cg Beclin1, Cg ATG5, Cg ATG16L, Cg LC3 and Cg The mRNA expression of P62 was significantly increased compared with the control group ( Figure 2 AE), were 6.01 times that of the control group ( p < 0.01), 3.23 times ( p < 0.01), 4.33 times ( p < 0.0001), 19.97 times ( p < 0.01) and 4.33 times ( p < 0.0001), indicating that ammonium chloride can promote the expression of autophagy-related genes in the D-type larvae; hematopoietic-related genes Cg Notch1, Cg TNF-2, Cg BMP-7, Cg CDC-6, Cg CDK-2 and Cg Runx were significantly increased compared with the control group ( Figure 2 FK), were 4.29 times that of the control group ( p < 0.05), 2.98 times ( p < 0.001), 4.09 times ( p < 0.0001), 2.67 times ( p <0.01), 2.22 times ( p < 0.01) and 3.38 times (p < 0.001), indicating that after ammonium chloride treatment of long oyster D-type larvae, the proliferation ability of blood lymphocytes was enhanced and the immunity of larvae was improved; antiviral related genes Cg TRIM, Cg Mx1 and Cg IFI44 was significantly increased compared with the control group ( Figure 2 LN), were 6.00 times that of the control group ( p < 0.001), 4.18 times ( p < 0.0001) and 4.07 times ( p < 0.01), indicating that ammonium chloride treatment can promote the immune response of D-type larvae to foreign viruses and improve the immunity of larvae; inflammation-related genes Cg IL17-1, Cg IL17-5 and Cg GSDME increased significantly compared with the control group, and was 2.42 times that of the control group ( p < 0.0001), 2.98 times ( p < 0.001) and 2.97 times ( p < 0.05) ( Figure 2 OQ), indicating that ammonium chloride can help D-type larvae quickly identify pathogens, activate antibacterial immune function, and thus improve immunity.
[0024] Experiment 3: Effects of experimental groups on the protein level of autophagy-related gene LC3 in D-type larvae of long oysters After the D-type larvae of the Example group of the present invention were treated with ammonium chloride for 24 h, samples of the D-type larvae of the control group and the Example group of the present invention were collected for protein extraction. The protein samples were directly stored at -80°C to detect the effect of ammonium chloride treatment on the autophagy-related protein LC3 of the long oyster D-type larvae.
[0025] First, the protein samples were quantified using Tubulin, and then the relative expression level of the autophagy-related protein LC3 was detected by Western blot after 24 h of ammonium chloride treatment in D-type larvae.
[0026] The specific operations are as follows: (1) To ensure the consistency of protein content in each group, the internal reference gene Tubulin was first quantified; (2) Cut out the desired SDS-PAGE gel area and cut the nitrocellulose membrane (NC membrane) and filter paper to the appropriate size; (3) According to the current direction of the semi-dry electrotransfer apparatus, place the 3 layers of filter paper, SDS-PAGE gel, NC membrane, and 3 layers of filter paper in the order from bottom to top. The current size = 1.2 × NC membrane area (cm 2) mA, time 1 h; (4) After the transfer is completed, add 5 mL of Western blot washing solution and wash the NC membrane three times on a fast shaking shaker for 5 minutes each time; (5) Soak the NC membrane in milk solution and shake slowly at room temperature for 2 h. Then, incubate 5 μL of Tubulin antibody with 5 mL of milk solution and shake slowly at room temperature for 3 h. (6) After adding the color developing solution, observe the experimental results using a chemiluminescence gel imaging system and save them to ensure that the amount of protein loaded in each group is the same; (7) After determining the amount of protein loaded in each group, the relative expression of the autophagy-related protein LC3 was detected using Western blot technology. The protein samples stored at -80°C were thawed on ice and then subjected to SDS-PAGE; (8) Cut out the desired SDS-PAGE gel region based on the protein size of LC3 and cut the nitrocellulose membrane (NC membrane) and filter paper of appropriate size; (9) According to the current direction of the semi-dry electrotransfer apparatus, place the 3 layers of filter paper, SDS-PAGE gel, NC membrane, and 3 layers of filter paper in the order from bottom to top. The current size = 1.2 × NC membrane area (cm 2 ) mA, time 25 min; (10) After the transfer is completed, add 5 mL of Western blot washing solution and wash the NC membrane three times on a fast shaking shaker for 5 minutes each time; (11) Soak the NC membrane in milk solution and shake slowly at room temperature for 2 h. Then, add 5 μL of LC3 antibody and 5 mL of milk solution and shake slowly at 4°C overnight. (12) Add 5 mL of Western blot washing buffer and wash the NC membrane three times with rapid shaking, each time for 5 minutes; (13) Add 5 μL horseradish peroxidase goat anti-rabbit and 5 mL milk solution and incubate at room temperature with slow shaking for 1 h; (14) Wash the NC membrane with Western blot washing buffer by rapid shaking three times, 5 min each time; (15) After adding the color developing solution, the experimental results were observed and saved using the chemiluminescence gel imaging system.
[0027] The experimental results are as follows Figure 3 The results showed that compared with the control group, the LC3-Ⅱ band of the D-type larvae in the Example group of the present invention was significantly enhanced after 24 hours of ammonium chloride treatment, indicating that the protein expression level of the autophagy-related protein LC3 increased, and the protein level was significantly correlated with the mRNA level ( Figure 2 D) consistent.
Claims
1. A method for cultivating long oyster seedlings capable of improving the immunity of type D larvae, characterized by: Fertilized eggs of Crassostrea gigas were obtained and cultured using artificial breeding methods. After the fertilized eggs developed into D-type larvae, the D-type larvae were treated with aquaculture water with a final concentration of ammonium chloride of 0.053 g / L for 24 h. The D-type larvae were then transferred to aquaculture water without ammonium chloride for continued culture.
2. The method for cultivating long oyster seedlings capable of improving the immunity of type D larvae according to claim 1, characterized in that: The method of treating the D-type larvae for 24 hours with a final ammonium chloride concentration of 0.053 g / L in aquaculture water comprises spraying an ammonium chloride solution into the aquaculture water of the early stage D-type larvae to achieve a final ammonium chloride concentration of 0.053 g / L, and aquaculture the D-type larvae for 24 hours.