Small cell piece treating agent for improving culture quality of fresh water pearls and treating method of small cell piece treating agent

Abstract

The invention discloses a small cell piece treating agent for improving the culture quality of fresh water pearls and a treating method thereof, and relates to the technical field of aquaculture. The small cell piece treating agent is prepared by taking a phosphoric acid buffer salt solution as a basic solution, and functional components comprise a double-antibiotic solution and various growth factors; after fresh water pearl mussel cell slices are prepared, the cell slices are soaked in the treating agent for 3-5 minutes, and then nucleus insertion and pearl culture are carried out. By adopting the treatment agent and the treatment method, the problem of poor antibacterial effect of the monoclonal antibody can be solved when the pearl culture production of the freshwater pearl mussels is carried out, postoperative infection is effectively inhibited, and wound healing is promoted; the technical problem that the pearl bag forming speed is low is solved, pearl bag forming is accelerated, and pearl quality is improved. The product and the method disclosed by the invention are suitable for preoperative treatment of small cell pieces in a freshwater pearl mussel nucleus insertion operation, and have the characteristics of low cost, high stability and good effect.

Description

Technical Field

[0001] The invention belongs to the field of aquaculture, and in particular relates to a cell flake treating agent and a treating method thereof for improving the cultivation quality of freshwater pearls. Background Art

[0002] Pearl nucleus insertion is a key step in artificial pearl culture. Freshwater pearl culture has high benefits because of its low investment, easy management and high returns. It is one of the agricultural characteristic industries and wealth-generating industries. Hypriosis cumingii ) is an important freshwater pearl oyster in my country. It is the first choice for freshwater pearl production because of its thick nacre, bright luster, fine and smooth texture and high-quality pearls. Modern artificial pearl cultivation technology is to implant small pieces of cells made from the mantle tissue of the donor pearl oyster into the recipient pearl oyster alone or together with the nucleus to form nucleus-free pearls or nucleus-containing pearls. The vitality of the small pieces of cells is an important factor affecting the quality of pearls.

[0003] Pearl oysters live in water for a long time, and their growth and development process are affected by the water quality environment. During the pearl cultivation process, pearl oysters need to obtain nutrients from the water to support the formation of pearls. Factors such as the temperature, pH value and nutrients of the aquaculture water will affect the growth of pearl oysters and the pearl formation process. However, during the nucleus insertion process, the cell fragments are affected by the osmotic pressure and pH of the water, resulting in an imbalance inside and outside the body, which leads to uneven quality of pearls formed in the pearl oysters. Therefore, maintaining a stable and good water quality environment during the pearl cultivation process is crucial to the formation of pearls.

[0004] The existing pearl cell flake treatment liquid usually contains some nutrients and antibacterial substances required for the growth of pearl oysters, such as chlortetracycline hydrochloride, chitosan, etc. These substances are mixed and concentrated with water in a certain proportion through relevant processes, and finally made into cell flake treatment liquid for pearl culture. It is soaked in the process of pearl oyster culture and nucleus insertion to ensure the growth needs of pearl oysters and to expel harmful bacteria and prevent postoperative infection. However, the pathogens in the current aquaculture water are changeable, and traditional reagents can no longer meet the antibacterial and growth requirements. The pearl oysters have a high pearl spitting rate, and the pearl particles produced are not full.

[0005] Growth factors are a class of proteins that can promote cell proliferation, differentiation, and repair, and have the effects of promoting wound healing, cell growth, and differentiation. Adding growth differentiation factor-11 in the treatment of diabetic wound healing can accelerate the wound healing rate; adding epidermal growth factor in the slow-release membrane reagent for healing wounds will have better antibacterial activity; adding a small molecule inhibitor of insulin-like growth factor-1 receptor can inhibit skeletal muscle hyperplasia; adding human fibroblast growth factor to oral gel has osteogenic induction effect. Using a sustained-release carrier to continuously release bFGF can continuously stimulate the regeneration of periodontal tissue. Nowadays, growth factors have been widely used in medical surgeries, but they have not been applied in pearl nucleation of freshwater pearl mussels.

[0006] Existing pearl-seeding cell piece treatment agents have certain limitations in production and formulation, and there is still a lack of a standardized treatment method for cell pieces. Problems such as low cell piece viability and serious bacterial contamination still cause partial rejection reactions of pearl mussels after inoculation, which in turn affects the formation of pearl sacs, the pearl seeding rate, and the quality of pearls. In view of this, the existing technology urgently needs to find a pearl cultivation method and a treatment agent used in the nucleation operation that can solve the rejection of the pearl nucleus by the mussel body, promote wound healing, improve the viability of cell pieces, and can meet mass operation requirements. Summary of the Invention

[0007] The purpose of the present invention is to solve the problems of high nucleus spitting rate and poor quality in the existing freshwater pearl cultivation. Based on the existing technology, the present invention is improved to provide a cell piece treatment agent and a treatment method for improving the quality of freshwater pearl cultivation.

[0008] The technical solution of the present invention is as follows: A cell piece treatment agent for improving the quality of freshwater pearl cultivation, selects phosphate buffered saline as the basic solution, the double-antibody component selects penicillin-streptomycin mixed solution, and the growth factors are composed of epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-1.

[0009] As a preferred embodiment, the treatment agent needs to add a 10% penicillin (100 U / ml)-streptomycin (0.1 mg / ml) mixed solution.

[0010] As a preferred embodiment, the treatment agent needs to add a mixed solution of 2 ng / mL epidermal growth factor, 15 ng / mL basic fibroblast growth factor, and 1 ng / mL insulin-like growth factor-1.

[0011] A treatment method for a cell piece treatment agent for improving the quality of freshwater pearl cultivation specifically includes the following steps: (1)Preparation of cell piece treatment agent: Add 10% penicillin (100 U / ml)-streptomycin (0.1 mg / ml) solution, 2 ng / mL epidermal growth factor, 15 ng / mL basic fibroblast growth factor, and 1 ng / mL insulin-like growth factor-1 to phosphate buffered saline solution, and uniformly mix the above nutrient tissue agents for standby; (2)Preparation and treatment of cell pieces: Obtain cell pieces with a size of 1×1 mm and a thickness of 0.5 - 0.8 mm according to the conventional operation of freshwater pearl nucleus insertion surgery. Immerse the cell pieces in the cell piece treatment agent obtained in step (1) for 3 - 5 minutes, and implant the soaked cell pieces and the pearl nucleus into the mantle body of the pearl oyster together with a through-piece needle; (3)Cultivation of pearl oysters: Place the pearl oysters with implanted pieces with the water outlet facing upwards and dry for 30 min - 1 h, then transfer them into a hanging net bag in the freshwater cultivation area for cultivation. Record the number of ejected nuclei and the viability of cell pieces. After 1 month of nucleus insertion and pearl cultivation, randomly sample each group to check the number of harvested pearls for pearl quality.

[0012] As a preferred embodiment, the cell piece treatment agent is prepared and used immediately, and the prepared cell piece treatment agent is used within 4 hours.

[0013] As a preferred embodiment, the three growth factors in step (1) need to be stored at -20°C for a long time.

[0014] As a preferred embodiment, the penicillin-streptomycin mixed solution, phosphate buffered saline solution, and the prepared cell piece treatment agent in step (1) need to be stored at 4°C for a long time.

[0015] As a preferred embodiment, insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and penicillin-streptomycin solution are all substances known in the prior art and can be purchased from the market.

[0016] The cell piece treatment agent and method for freshwater pearl oysters of the present invention take the formation of pearl sac, pearl quality, etc. as basic parameters, introduce the concept of comprehensive evaluation index, quantify the pearl cultivation effect, improve the activity of cell pieces, the affinity between pearl nucleus and pieces, and effectively inhibit bacterial infection during the operation.

[0017] Compared with the prior art, the pearls obtained after the treatment of the present invention have the following beneficial effects: 1. Adding penicillin-streptomycin solution to the cell piece treatment agent to treat cell pieces can effectively inhibit bacterial infection, enhance the immune response of pearl oysters, and accelerate the wound healing caused by nucleus insertion surgery.

[0018] 2. By treating the cell pieces with human insulin-like growth factor-1, epidermal growth factor, and basic fibroblast growth factor, the viability of the cell pieces is effectively improved, and the growth of the pearl sac is accelerated.

[0019] 3. The selected phosphate buffer solution has a salt balance and an adjustable suitable pH buffering effect, ensuring that the overall pH of the cell piece treatment solution is between 7 and 8. While cleaning the surface of the cell pieces, the cell piece treatment agent is more adapted to the internal environment of the pearl oyster. The prepared solution and related nutrients can be better absorbed by the pearl oyster, reducing the occurrence of rejection reaction. The pearl formation rate is increased by 1.2% compared with the prior art.

[0020] 4. Before implanting the cell pieces into the pearl oyster, soak them in the treatment agent of the present invention for 3 - 5 minutes, which can promote the secretion of nacre, and the nacre layer thickness is increased by 0.2 mm compared with the prior art.

[0021] 5. Using the cell piece treatment agent and treatment method of the present invention can improve the quality of pearls. And this method is easy to operate, with obvious effects, and can be used for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the structure after nucleus insertion of Hyriopsis cumingii in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The following will describe the embodiments of the present invention in detail with reference to the drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0024] Taking the cultivation of freshwater pearls with Hyriopsis cumingii as an example, the specific implementation steps of the experimental group are as follows: (1) Selection and temporary cultivation of the donor mussel and the pearl oyster: Select 1-year-old healthy and intact Hyriopsis cumingii as the donor mussel, and select 2-year-old healthy and intact Hyriopsis cumingii as the pearl oyster. The selected pearl oysters and donor mussels are first placed in a clear water pond for temporary cultivation for 30 minutes to 2 hours, and at the same time, oxygen circulation is carried out to keep the oxygen concentration at 5 - 7 mg / L.

[0025] (2) Preparation of the cell piece treatment agent: Add the treatment agent of the present invention. Taking the preparation of 50 ml of the treatment agent of the present invention as an example: Add 5 mL of penicillin (100 U / ml)-streptomycin (0.1 mg / ml) solution, 100 ng of human epidermal growth factor, 750 ng of human basic fibroblast growth factor, and 50 ng of human insulin-like growth factor-1 to the phosphate buffered saline solution, mix evenly, and add to 50 mL with the phosphate buffered saline solution for standby.

[0026] (3)Obtaining cell pieces: Inside the color line of the mantle epithelial tissue of the donor mussel in Step 1, make a smooth, firm, and decisive cut with even force, reaching the middle connective tissue layer of the mantle to obtain the entire mantle tissue; starting from the front of the obtained mantle tissue with forceps, tear the mantle tissue to get the outer membrane of the mantle, and remove the connective tissue to obtain cell pieces; use a microtome knife to cut the cell pieces into a size of 1×1 mm and a thickness of 0.5 - 0.8 mm.

[0027] (4)Treatment and implantation of cell pieces: Place the cell pieces cut in Step 3 in the cell piece treatment agent prepared in Step 2 and completely immerse them for 3 - 5 minutes; select round beads with a diameter of 2.00 mm, with a smooth, white, and crack - free or non - flat head surface; use a through - piece needle to implant the soaked cell pieces and the beads together into the right mantle body of the pearl - producing mussel in Step 1 (as Figure 1 ).

[0028] (5)Culturing pearl - producing mussels: Place the pearl - producing mussels with implanted pieces with the water outlet facing upwards and dry them for 30 min - 1 h, then transfer them into a hanging net bag in the fresh - water aquaculture area for culturing. Record the number of nucleus - spitting and the viability of cell pieces. Randomly sample and check the number of harvested pearls for the pearl quality 1 month, 2 months, 3 months, and 5 months after nucleus - insertion. Determine the intensive culture method for freshwater pearl mussels to improve the pearl yield and quality.

[0029] Taking the cultivation of freshwater pearls with Hyriopsis cumingii as an example, the commonly used method in the current base is used as the control group, and its specific implementation steps are as follows: A cell piece treatment agent for improving the quality of freshwater pearl cultivation and its treatment method. The difference in the method from the specific implementation steps of the experimental group lies in the preparation of the cell piece treatment agent in Step 2. The treatment agent is a phosphate - buffered saline solution added with an appropriate amount of chlortetracycline hydrochloride. Use a through - piece needle to implant the soaked cell pieces and the beads together into the left mantle body of the pearl - producing mussel in Step 1 (as shown in Table 1).

[0030] The statistical results of the pearl quality cultivated by the control group and the experimental group are shown in Table 1.

[0031]

[0032] Note: The identification and evaluation of pearl luster and smoothness levels are mainly based on the national standard "Pearl Grading" (GB / T 18781 - 2008).

[0033] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A cell flake treating agent for improving the quality of freshwater pearl cultivation, characterized in that: Phosphate buffered saline solution was selected as the basic solution, penicillin-streptomycin mixed solution was selected as the double antibody component, and the growth factor was prepared from epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-1 and.

2. A cell flake treating agent for improving the quality of freshwater pearl cultivation according to claim 1, characterized in that: The treatment agent needs to be added with 10% penicillin-streptomycin mixed solution.

3. The cell flake treating agent for improving the quality of freshwater pearl cultivation according to claim 1, characterized in that: The treatment agent needs to add a mixed solution of 2 ng / mL epidermal growth factor, 15 ng / mL basic fibroblast growth factor, and 1 ng / mL insulin-like growth factor-1.

4. A method for treating a cell flake treating agent for improving the quality of freshwater pearl cultivation as claimed in claim 1, comprising the following steps: (1) Preparation of cell piece treatment agent: add 10% penicillin-streptomycin solution, 2 ng / mL epidermal growth factor, 15 ng / mL basic fibroblast growth factor, and 1 ng / mL insulin-like growth factor-1 to phosphate buffered saline, mix the above nutrient tissue agents evenly and set aside; (2) Preparation and treatment of cell pieces: According to the freshwater pearl nucleus insertion operation, obtain cell pieces with a size of 1×1 mm and a thickness of 0.5-0.8 mm, soak the cell pieces in the cell piece treatment agent obtained in step (1) for 3-5 minutes, and implant the soaked cell pieces and the pearl nucleus into the mantle of the pearl oyster with a needle; (3) Pearl oyster culture: After the pearl oysters are implanted, place them in a dry place with the water outlet facing upward for 30 minutes to 1 hour, and then move them into a net bag hung in the freshwater culture area for culture. The number of pearls ejected and the vitality of the cell fragments are recorded. One month after the pearls are implanted, each group is randomly sampled to check the quality of the pearls and the number of pearls collected.

5. The method for treating a cell flake treating agent for improving the quality of freshwater pearl cultivation according to claim 4, characterized in that: The cell flakes treating agent is prepared and used immediately, and the prepared cell flakes treating agent can be used within 4 hours.

6. The method for treating a cell flake treating agent for improving the quality of freshwater pearl cultivation according to claim 4, characterized in that: The three growth factors in step (1) need to be stored at -20°C for a long time.

7. The method for treating a cell flake treating agent for improving the quality of freshwater pearl cultivation according to claim 4, characterized in that: The penicillin-streptomycin mixed solution, phosphate buffered saline solution and the prepared cell piece treating agent in step (1) need to be stored at 4° C. for a long time.

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