A cell flake treating agent for improving the quality of freshwater pearl cultivation and a treating method thereof

Abstract

The present invention discloses a cell flake treatment agent and a treatment method thereof for improving the quality of freshwater pearl cultivation, and relates to the field of aquaculture technology. The cell flake treatment agent is prepared with a phosphate buffered saline solution as the base solution, and the functional components include a double antibiotic solution and a plurality of growth factors; after preparing the freshwater pearl oyster cell flakes, the cell flakes are soaked in the treatment agent of the present invention for 3-5 minutes, and then the nucleus is inserted into the pearls. Using the treatment agent and treatment method of the present invention to produce freshwater pearl oyster pearls can overcome the problem of poor monoclonal antibody antibacterial effect, effectively inhibit postoperative infection, and promote wound healing; overcome the technical difficulty of slow pearl sac formation rate, accelerate pearl sac formation and improve pearl quality. The product and method of the present invention are suitable for preoperative treatment of cell flakes in freshwater pearl oyster nucleus insertion surgery, 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 particularly 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 cultivation. Freshwater pearl cultivation has high efficiency because of its low investment, easy management and high returns. It is one of the agricultural specialty industries and one of the wealth-generating industries. Hypriosis cumingii The oyster is a key freshwater pearl-cultivating oyster in my country. Its thick, brightly colored, smooth, and finely polished nacre, resulting in high-quality pearls, makes it the preferred choice for freshwater pearl cultivation. Modern artificial pearl cultivation involves implanting small cell fragments made from the mantle tissue of a donor pearl oyster, either alone or with a nucleus, into a recipient pearl oyster to create either nucleus-free or nucleated pearls. The vitality of these cell fragments is a crucial factor influencing pearl quality.

[0003] Pearl oysters live in water for extended periods, and their growth and development are influenced by the water quality. During the pearl cultivation process, pearl oysters require nutrients from the water to support pearl formation. Factors such as the temperature, pH, and nutrients of the culture water influence the growth and pearl formation process. However, during the nucleus insertion process, the cell fragments are affected by the osmotic pressure and pH of the water, leading to an imbalance between the internal and external environment. This results in varying quality of the pearls formed within the pearl oysters. Therefore, maintaining a stable and good water quality environment during the pearl cultivation process is crucial for pearl formation.

[0004] Existing pearl cell flake processing solutions typically contain nutrients and antibacterial substances required for the growth of pearl oysters, such as chlortetracycline hydrochloride and chitosan. These substances are mixed and concentrated with water in a certain proportion through a related process to create a cell flake processing solution for pearl cultivation. This solution is then soaked in the pearl oysters during the cultivation and nucleus insertion process to ensure their growth needs, eliminate harmful bacteria, and prevent post-operative infection. However, the current aquaculture waters are plagued by a variety of pathogens, and traditional reagents no longer meet the antibacterial and growth requirements. As a result, the pearl oysters have a high pearling rate, and the resulting pearls are not full.

[0005] Growth factors are a class of proteins that promote cell proliferation, differentiation, and repair, and they have the potential to promote wound healing, cell growth, and differentiation. Adding growth differentiation factor-11 to diabetic wound healing can accelerate wound healing; adding epidermal growth factor to sustained-release membranes for wound healing enhances antimicrobial activity; adding small molecule inhibitors of the insulin-like growth factor-1 receptor can inhibit skeletal muscle proliferation; human fibroblast growth factor added to oral gels has an osteoinductive effect, and using sustained-release carriers to continuously release bFGF can stimulate periodontal tissue regeneration. Growth factors are widely used in medical procedures today, but they have not yet been used in freshwater pearl oyster nucleus insertion for pearl cultivation.

[0006] Existing treatment agents for pearl-cultivating cell flakes have certain limitations in production and formulation, and there is a lack of standardized treatment methods for treating cell flakes. Problems such as low cell flake viability and severe bacterial contamination can still cause some rejection reactions in pearl-cultivating oysters after inoculation, thereby affecting pearl sac formation, pearl cultivation rate, and pearl quality. Given this, there is an urgent need to find a pearl cultivation method and treatment agent for nucleus insertion that can address the oyster's rejection of the nucleus, promote wound healing, increase cell flake viability, and enable large-scale operations. Summary of the Invention

[0007] The present invention aims to solve the problems of high nucleus ejection rate and poor quality in existing freshwater pearl cultivation. Based on the existing technology, the present invention provides a cell flake treatment agent and treatment method for improving the quality of freshwater pearl cultivation.

[0008] The technical solutions of the present invention are as follows:

[0009] A cell flake treatment agent for improving the quality of freshwater pearl cultivation is prepared by selecting a phosphate buffered saline solution as a base solution, selecting a penicillin-streptomycin mixed solution as a double-antibody component, and selecting growth factors from epidermal growth factor, basic fibroblast growth factor, and insulin-like growth factor-1.

[0010] As a preferred embodiment, the treatment agent needs to be added with a 10% penicillin (100 U / ml)-streptomycin (0.1 mg / ml) mixed solution.

[0011] As a preferred embodiment, the treatment agent needs to be added with 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.

[0012] A method for treating a cell flake treatment agent to improve the quality of freshwater pearl cultivation comprises the following steps:

[0013] (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, mix the above nutrient tissue agents evenly and set aside;

[0014] (2) Preparation and treatment of cell flakes: According to the routine operation of freshwater pearl nucleus insertion surgery, obtain cell flakes with a size of 1×1 mm and a thickness of 0.5-0.8 mm, soak the cell flakes in the cell flake treatment agent obtained in step (1) for 3-5 minutes, and use a needle to implant the soaked cell flakes and the pearl nucleus into the mantle of the pearl oyster;

[0015] (3) Pearl oyster cultivation: Place the pearl oysters with the water outlet facing upwards and dry for 30 minutes to 1 hour after implantation, then move them into the net bag hanging in the freshwater aquaculture area for cultivation. Record the number of nuclei ejected and the vitality of the cell fragments. After one month of nuclei implantation, each group randomly samples the pearls to check the quality and number of pearls collected.

[0016] As a preferred embodiment, the cell flakes treating agent is prepared and used immediately, and the prepared cell flakes treating agent can be used within 4 hours.

[0017] As a preferred embodiment, the three growth factors in step (1) need to be stored at -20°C for a long time.

[0018] As a preferred embodiment, 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 term.

[0019] As a preferred embodiment, the 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.

[0020] The cell flake processing agent and method of the freshwater pearl oyster of the present invention takes the pearl sac formation, pearl quality and the like as basic parameters, introduces the concept of comprehensive evaluation index, quantifies the pearl cultivation effect, improves the activity of the cell flakes, the affinity between the bead nucleus and the flakes, and effectively inhibits bacterial infection during surgery.

[0021] Compared with the prior art, the pearls obtained after treatment by the present invention have the following beneficial effects:

[0022] 1. Adding penicillin-streptomycin solution to the cell fragment treatment agent to treat the cell fragments can effectively inhibit bacterial infection, enhance the immune response of pearl oysters, and accelerate the healing of wounds caused by nuclear insertion surgery.

[0023] 2. By adding human insulin-like growth factor-1, epidermal growth factor, and basic fibroblast growth factor to treat the cell flakes, the vitality of the cell flakes can be effectively improved and the growth of the pearl sac can be accelerated.

[0024] 3. The selected phosphate buffer has salt balance and adjustable pH buffering effect, ensuring that the overall pH of the cell flake treatment solution is between 7 and 8. While cleaning the surface of the cell flakes, the cell flake treatment agent makes it more adaptable to the internal environment of the pearl oyster. The configured solution and related nutrients can be better absorbed by the pearl oyster, reducing the occurrence of its rejection reaction. Its pearl formation rate is increased by 1.2% compared with the existing technology.

[0025] 4. Before the cell pieces are implanted into the pearl oyster, they are first soaked in the treatment agent of the present invention for 3-5 minutes, which can promote the secretion of nacre and increase the thickness of the nacre layer by 0.2 mm compared with the original technology.

[0026] 5. The cell flake treating agent and treating method of the present invention can improve the quality of pearls. The method is easy to operate, has obvious effects, and can be used for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the structure of the cumingii clam after the nucleus is inserted in one embodiment of the present invention. DETAILED DESCRIPTION

[0028] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0029] Taking the cultivation of freshwater pearls by Hyriopsis cumingii as an example, the specific implementation steps of the experimental group are as follows:

[0030] (1) Selection and temporary rearing of supply clams and pearl oysters: Select healthy, intact 1-year-old Hyriopsis cumingii as supply clams, and select healthy, intact 2-year-old Hyriopsis cumingii as pearl oysters. The selected pearl oysters and supply clams are first placed in a clean water pool for temporary rearing for 30 minutes to 2 hours, while oxygen circulation is carried out to maintain the oxygen concentration at 5-7 mg / L.

[0031] (2) Preparation of cell piece treatment agent: Add the treatment agent of the present invention. Take 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 phosphate buffered saline solution, mix evenly, and add phosphate buffered saline solution to 50 ml for later use.

[0032] (3) Obtaining cell flakes: Within the color line of the mantle epithelial tissue of the clams provided in step 1, make a cut evenly and decisively, reaching the depth of the middle connective tissue layer of the mantle to obtain the entire mantle tissue; use tweezers to tear the mantle tissue from the front of the obtained mantle tissue to obtain the outer membrane of the mantle, remove the connective tissue to obtain cell flakes; use a microtome to cut the cell flakes into pieces with a size of 1×1mm and a thickness of 0.5-0.8mm.

[0033] (4) Treatment and implantation of cell flakes: Place the cell flakes cut in step 3 in the cell flake treatment agent prepared in step 2 and completely immerse them for 3-5 minutes; select 2.00mm round particles as bead nuclei, with smooth and white nuclei without cracks or flat heads; use a needle to implant the soaked cell flakes and bead nuclei into the right mantle of the pearl oyster in step 1 (such as Figure 1 ).

[0034] (5) Pearl oyster culture: Place the pearl oysters with the water outlet facing upwards and dry for 30 minutes to 1 hour after planting, then move them into the net bag hanging in the freshwater culture area for culture, record the number of nuclei spitted out and the vitality of the cell fragments, and randomly sample the pearls to check the quality of the pearls after 1 month, 2 months, 3 months, and 5 months of nuclei culture, and determine the intensive culture method of freshwater pearl oysters to improve the pearl yield and quality.

[0035] Taking the cultivation of freshwater pearls by Hyriopsis cumingii as an example, the commonly used method at the base is the control group, and its specific implementation steps are as follows:

[0036] A cell flake treating agent and a treating method for improving the quality of freshwater pearl cultivation are disclosed. The method differs from the specific implementation steps of the experimental group in that the cell flake treating agent is prepared in step 2. The treating agent is a phosphate buffered saline solution with an appropriate amount of chlortetracycline hydrochloride added. The soaked cell flakes and pearl nuclei are implanted into the left mantle of the pearl oyster in step 1 together with a needle (as shown in Table 1).

[0037] The statistical results of the pearl quality of the control group and the experimental group are shown in Table 1.

[0038]

[0039] Note: The evaluation of pearl luster and smoothness level is mainly based on the national standard "Pearl Grading" (GB / T 18781-2008).

[0040] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for treating freshwater pearls with a cell flake treatment agent to improve the quality of freshwater pearl cultivation, characterized in that: The specific steps include: (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 flakes: According to the routine operation of freshwater pearl nucleus insertion surgery, obtain cell flakes with a size of 1×1 mm and a thickness of 0.5-0.8 mm, soak the cell flakes in the cell flake treatment agent obtained in step (1) for 3-5 minutes, and use a needle to implant the soaked cell flakes and the pearl nucleus into the mantle of the pearl oyster; (3) Pearl oyster cultivation: Place the pearl oysters with the water outlet facing upwards and dry for 30 minutes to 1 hour after implantation, then move them into the net bag hanging in the freshwater aquaculture area for cultivation. Record the number of nuclei ejected and the vitality of the cell fragments. After one month of nuclei implantation, each group randomly samples the pearls to check the quality and number of pearls collected.

2. The method for treating freshwater pearls with a cell flake treating agent for improving the quality of freshwater pearl cultivation according to claim 1, characterized in that: The cell flake treatment agent is prepared and used immediately, and the prepared cell flake treatment agent can be used within 4 hours.

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

4. The method for treating freshwater pearls with a cell flake treating agent for improving the quality of freshwater pearl cultivation according to claim 1, 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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