CN113317245B - Method for regulating and controlling generation of pearl sac of pearl-breeding mussel and method for producing pearl - Google Patents
Method for regulating and controlling generation of pearl sac of pearl-breeding mussel and method for producing pearl Download PDFInfo
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- CN113317245B CN113317245B CN202110740689.1A CN202110740689A CN113317245B CN 113317245 B CN113317245 B CN 113317245B CN 202110740689 A CN202110740689 A CN 202110740689A CN 113317245 B CN113317245 B CN 113317245B
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/54—Culture of aquatic animals of shellfish of bivalves, e.g. oysters or mussels
- A01K61/56—Culture of aquatic animals of shellfish of bivalves, e.g. oysters or mussels for pearl production
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention relates to a method for inducing pearl cultivation, in particular to a method for regulating and controlling generation of pearl sacs of pearl mussels and a method for producing pearls. The technical scheme provided by the invention is to promote and improve the generation of the pearl sac by reducing the oxidative stress level of the pearl mussel after the nucleus planting or the sheet planting operation. Compared with the prior art in which the main influence of the regulation and control mode is the growth speed of the pearl sac, the invention not only accelerates the growth speed of the pearl sac, but also realizes regulation and control of the shape of the pearl, thereby realizing regulation and control of the early shape of the pearl, solving the problem of regulation and control of the shape of the pearl and providing possibility for manually controlling the appearance of the pearl.
Description
Technical Field
The invention relates to a method for inducing pearl cultivation, in particular to a method for regulating and controlling generation of pearl sacs of pearl cultivators and a method for producing pearls.
Background
Pearl is a valuable organic jewelry formed by the secretion of nacre from the pearl sac of shellfish. The quality of a pearl, such as size, shape, color, luster, and finish, is a major factor in determining the value of a pearl. China is a big country for artificially culturing pearls, however, the pearl culture industry in China, particularly the proportion of high-quality commercial pearls of fresh water pearls (about 10 percent) is low, not only about 5 percent of the total world yield is obtained (FAO,2018), but also the production mode of obtaining a small amount of high-quality pearls at high yield through large-area culture occupies valuable culture water areas and other resources, and the economic, ecological and social benefits are low.
The artificially cultured pearl is cultured by implanting small pieces of mantle tissue into a pearl sac formed by the development of pearl-breeding shells (mussels), and the structure and the secretion capacity of the pearl sac are key factors and bases influencing the quality of the pearl.
In the prior art, the regulation and control method for the development of the pearl sac of the pearl culturing shell (mussel) is mainly realized by inhibiting the immunological rejection capability of the pearl culturing shell (mussel), enhancing the immunity and the calcium ion metabolic capability of the pearl culturing shell (mussel) and the like. However, little is known about methods for improving the development of the pearl sac by modulating the oxidative stress in pearl mussels after nucleus or patch implantation.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a method for regulating and controlling the production of pearl sacs of pearl mussels.
The second purpose of the invention is to provide a method for producing high-quality pearls by using pearl mussels.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method of regulating the production of pearl sacs in pearl mussels, the method comprising reducing the level of oxidative stress in the pearl mussels following a nucleus or patch grafting operation.
Further, the level of oxidative stress is reduced by inhibiting the pro-phenoloxidase system ROS production pathway.
Further, the method for inhibiting the pro-phenoloxidase system ROS production pathway comprises activating the pearl mussel antioxidant enzyme system or inhibiting the enzymatic activity of phenoloxidase with a phenoloxidase inhibitor using a pathogen pattern recognition molecule.
Further, the pathogen pattern recognition molecule comprises lipopolysaccharide, peptidoglycan, lipoteichoic acid, mannan, dextran or mannose, preferably lipopolysaccharide;
preferably, the phenol oxidizing enzyme inhibitor comprises 1-phenyl-2-thiourea, 4-hexylresorcinol or kojic acid;
preferably, the lipopolysaccharide activation method comprises: injecting lipopolysaccharide at the muscle of the adductor muscle at a dose of 0.1-0.3 μ g/g mussel weight within 0-6 days after nucleus or patch implantation operation of the pearl mussel.
Further, the level of oxidative stress is reduced by antioxidants;
preferably, the antioxidant comprises vitamin C, water-soluble vitamin E, D-alpha-tocopheryl polyethylene glycol succinate, taurine or a rare earth element; preferably taurine or a rare earth element.
Further, methods of taurine inhibition include: feeding the pearl mussel with bait containing 0.5-1.5 w/v% taurine 0-40 days after nucleus planting or sheet planting operation, 3 times per day; preferably 0 to 30 days;
preferably, the bait comprises 2-4g/L chlorella, 6.5-8.5g/L soybean milk and 5-15mg/ml yeast.
Further, the method for inhibiting rare earth elements comprises the step of performing 10 days after nucleus or implant operation of pearl mussel -2 -10 -1 Mu mol/only adductor muscle is injected with rare earth element cerium.
Further, the temperature of the culture water body for the pearl mussel is 25-29 ℃.
Further, the pearl mussel is a hyriopsis cumingii; the preferred is hyriopsis cumingii with 1-year-old shell length of 8-10 cm.
A method for producing pearl from pearl culturing mussel comprises culturing pearl sac in the above method, and culturing the pearl mussel containing pearl sac to obtain pearl.
Compared with the prior art, the invention has the beneficial effects that:
the technical scheme provided by the invention is that the generation of the pearl sac is promoted and improved by reducing the oxidative stress level of the pearl mussel after the nucleus or the sheet grafting operation. The inventor finds out through experimental research that, in the process of forming the pearl sac of the pearl-cultivating mussel after the nucleus planting or the sheet planting operation, the reduction of the oxidative stress level of the pearl-cultivating mussel can promote the formation of the pearl sac, effectively improve the pearl deposition and improve the roundness of the pearl, thereby improving the yield and the quality of the pearl. The method only intervenes in the formation process of the pearl sac, and the original pearl cultivation conditions are little changed, the cost is low, and the practical range is wide. Compared with the prior art in which the regulation and control mode mainly influences the growth speed of the pearl sac, the method has the advantages that the growth speed of the pearl sac is accelerated, the shape of the pearl sac is regulated and controlled, the early shape of the pearl is regulated and controlled, the problem of regulation and control of the shape of the pearl is solved, and the possibility of manually controlling the shape of the pearl is provided.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a comparison of the morphology of the pearl sacs at different concentrations of LPS treatment 6d and 12d in example 1;
FIG. 2 is a graph showing the difference in the level of Reactive Oxygen Species (ROS) in the hemolymphocytes of pearl mussel (row A) and the pearl sac (row B) at 6d after LPS treatment at different concentrations in example 1;
FIG. 3 is a comparison of the levels of indicators related to oxidative stress of the pearl sac at 6 and 12d after LPS treatment at different concentrations in example 1;
FIG. 4 is a graph showing a comparison of relative expression changes of the SR gene of scavenger receptor at 6 and 12d after LPS treatment at different concentrations in example 1;
FIG. 5 is a microscopic observation image of the morphology of the pearl sac tissue of hyriopsis cumingii 6 days after the implantation operation in example 2;
FIG. 6 is a morphological microscopic observation picture of pearl sac tissue of hyriopsis cumingii 12 days after the operation of the implant in example 2;
FIG. 7 is a graph showing the difference in the levels of ROS and superoxide anion free radicals in blood cells of pearl mussel treated with different concentrations of rare earth cerium in example 2;
FIG. 8 is a comparison of the characteristics of the redox indicators of the tissues of the pearl sac after the treatment of different concentrations of CeCl3 drop tablets for 6 days in example 2;
FIG. 9 is a scanning electron microscope image of the nacre platelets treated with different solutions for 12 days in example 2;
FIG. 10 shows the variation of the oxidative stress related index and the pearl substance deposition amount of the pearl mussel fed with different amounts of taurine in the bait according to example 3.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
The invention provides a method for regulating and controlling the generation of pearl sacs of pearl-breeding mussels, which can promote and improve the generation of the pearl sacs by reducing the oxidative stress level of the pearl-breeding mussels after nucleus or sheet grafting operation. The inventor finds out through experimental research that in the process of forming the pearl sac of the pearl-culturing mussel after nucleus planting or sheet planting operation, if the oxidative stress level of hemolymph and tissue slices/the pearl sac of the pearl-culturing mussel is reduced, the formation of the pearl sac can be promoted, the pearl sedimentation speed is effectively increased, the roundness of the pearl is improved, and the yield and the quality of the pearl are improved. The method only intervenes in the pearl sac forming process, and the original pearl cultivation conditions are little changed, the cost is low, and the practical range is wide.
It should be noted that the core implantation or the implantation in the present invention may be performed by a conventional method commonly used in the art, and is not particularly limited herein.
In order to reduce the oxidative stress response of the pearl mussel, the inventor finds out through experiments that the reduction can be realized by inhibiting the ROS generation pathway of the prophenoloxidase system of the pearl mussel. Specifically, the oxidative stress level of pearl mussels can be reduced by activating the pearl mussel antioxidant enzyme system or inhibiting the enzymatic activity of phenol oxidase by using a pathogen pattern recognition molecule. Wherein the pathogen pattern recognition molecule may be Lipopolysaccharide (LPS), Peptidoglycan (PGN), lipoteichoic acid (LTA), mannan (LAM), dextran (Glucan) or mannose (Man), and preferably is lipopolysaccharide; the phenol oxidase inhibitor includes 1-phenyl-2-thiourea (1-phenyl-2-thiourea, PTU), 4-hexylresorcinol (4-hexylresorcinol,4-HR) or Kojic acid (Kojic acid).
In one embodiment, the lipopolysaccharide is used for activation, and the specific method can be that the injection is carried out on the adductor muscle of the pearl mussel according to the dosage of 0.1-0.3 mu g of lipopolysaccharide added per gram of the weight of the pearl mussel within 0-6 days after the nucleus or the sheet grafting operation of the pearl mussel. It should be noted that the injection of lipopolysaccharide may be performed simultaneously with the core implantation or the implantation, may be performed separately, and may be completed within 0 to 6 days after the operation, preferably simultaneously, to reduce the cost.
In addition to inhibiting the pro-phenoloxidase system ROS production pathway to achieve a reduction in oxidative stress levels, reduction in oxidative stress levels in pearl mussels can also be achieved through antioxidant inhibition. Specifically, the antioxidant can be vitamin C, water-soluble vitamin E, D-alpha-vitamin E polyethylene glycol succinate (TPGS), taurine or rare earth elements. Among them, taurine and a rare earth element are preferable.
In one embodiment, the method for inhibiting with taurine comprises feeding the pearl mussel with a bait containing 0.5-1.5 w/v% taurine 3 times per day within 0-40 days or 0-30 days after the nucleus planting or the sheet planting operation. Wherein the bait is preferably 2-4g/L chlorella, 6.5-8.5g/L soybean milk and 5-15mg/ml yeast, and 0.5-1.5 w/v% is 0.5-1.5g taurine per 100ml bait.
In one embodiment, the method for inhibiting by using rare earth elements comprises performing the inhibition according to 10 days within 0-6 days after nucleus or implant operation of pearl mussel -2 -10 -1 Mu mol/only adductor muscle is injected with rare earth element cerium. Preferably CeCl 3 。
In the process of producing the pearl sac, the culture temperature may be, but not limited to, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 29 ℃.
The pearl sac generation method provided by the invention is particularly suitable for the hyriopsis cumingii which is a hyriopsis cumingii, in particular to the hyriopsis cumingii with the 1-year-old shell length of 8-10 cm.
The invention also provides a method for producing pearls by the pearl mussels, and the pearls can be obtained by culturing the pearl mussels by a conventional method after the pearl sacs are generated by the method.
In the above embodiment, when the pearl mussel is treated with lipopolysaccharide or rare earth element, the pearl mussel is required to be cultured at 25-29 ℃ within 0-6 days, and then the pearl sac-forming pearl mussel is cultured in a conventional manner. When taurine is used for treating pearl mussels, the pearl mussels need to be cultivated at 25-29 ℃ within 0-40 days or 0-30 days, and then the pearl mussels with pearl sacs are cultivated in a conventional mode.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1LPS stimulation to modulate the Prophenolic oxidase System and the level of oxidative stress in Pearl mussels to promote the development of the Pearl sacs and pearl production
1.1 Experimental materials
Selecting 1-year-old healthy hyriopsis cumingii with a body length of 8-10cm as a wafer supply mussel and a pearl breeding mussel, and performing a wafer grafting operation according to a conventional method: the pallium tissue small piece with the size of 3.0x 3.0mm is manufactured by adopting a tearing method. 12 pieces of tissue slices are respectively implanted into the marginal membranes at the two sides of the mantle of the pearl mussel.
1.2LPS injection treatment
While the implants were performed, LPS was injected into the adductor muscle, and the test was performed by injecting 100. mu.l of PBS (0.01M) as a control group (PBS control group for short) and injecting medium-concentration and high-concentration LPS solutions (final LPS concentration of the pearl mussel is 0.2 and 0.4. mu.g LPS/g mussel weight) as test groups (medium-concentration group and high-concentration group for short), and 60 pearl mussels were repeated per group. Placing the injected pearl mussel in a greenhouse with water temperature of 26-28 deg.C for culturing, and hanging with net cage at water depth of 40 cm. After 6 and 12 days of cultivation, 3 pearl sacs of the pearl mussels are separated and fixed by 4 percent paraformaldehyde for preservation, and the morphological histochemical analysis of the pearl sacs is carried out.
1.3 Reactive Oxygen Species (ROS) assay
Internal O of blood cells and pearl sac tissue 2 – Horizontal and removal of O 2 – External ROS levels were measured on a flow cytometer (BD Accuri C6) using the Dihydroethidium (DHE) method and the 2', 7' -dichlorofluorescein (DCFH-DA) method, respectively, and the active oxygen content was measured as mean fluorescence intensity (Geo mean). The test of the pearl sacs which develop for 6d and 12d under the treatment of LPS injection with different concentrations after the transplantation of the plant is carried out, the result is shown in figure 2, and the test results are as follows: geometric mean representation of DCFH fluorescence Total intensity, significance of difference P<0.05, and the number n of the pearl oysters is 5. The results showed that at 6d after the implantation, the ROS levels of the pearl sac tissues in the medium concentration group were lower than those in the PBS control group and the high concentration group, compared to the PBS control group (row B of fig. 2). LPS treatment in blood lymphocytes ROS levels in the large granular, small granular and clear cells groups were significantly lower than in the PBS control group (P)<0.05) (line a of fig. 2).
1.4 histochemical analysis
Taking out 4 pearl sacs of pearl mussels from each treatment group respectively on days 6 and 12 after the operation of the grafting, and fixing the pearl sacs by using paraformaldehyde solution; preparing a tissue sample into a tissue loading piece, embedding, slicing, dewaxing and HE dyeing, and observing the structure of the pearl sac tissue by using a Nikon 80i fluorescence microscope. The results are shown in FIG. 1, in which (a) is the comparison of the morphology of the pearl sac when different concentrations of LPS are treated for 6 d; (b) the figure shows the comparison result of the pearl sac shape when the LPS is treated for 12d at different concentrations. In the graphs (a) and (b), (A-C) are the pearl sac forms of 6d and 12d after the pearl mussel growing in PBS control group (0. mu.g/g), medium concentration LPS experimental group (0.2. mu.g/g) and high concentration LPS experimental group (0.4. mu.g/g), respectively. The black boxes indicate the areas of the magnified display, and (D-F) are high magnification views of the local areas in the panels (A-C), respectively. S: tissue patch, IS: pearl sac space, CT: connective tissue, M: muscle tissue, OEC: epithelial cells outside the mantle, IEC: epithelial cells inside the mantle, H: hemolymphocytes, EC: tissue platelet epithelial cells, HD: blood cell debris, CD: nacre deposition, PSC: the pearl bag cavity. The scale bar in the panel was 500 μm (A-C), 100 μm (E-G). The number n of the pearl sacs is 4.
Histochemical analysis results show that the degree of fold of the inner skin of the pearl sac of the medium concentration group (0.2 mug/g LPS) is relatively lower at 6d after the injection treatment compared with the PBS control group and the high concentration group (0.4 mug/g LPS), the inner skin cells are vigorous in division, the shapes are more flame-shaped, the cells are longer, but the length difference does not reach the obvious difference level (a) of a graph in a figure 1); furthermore, the number of blood lymphocytes in the pearl sac increases with increasing LPS concentration.
At 12d after injection treatment, the tissue slices of the medium-concentration group are fused with mantle tissue of the pearl mussel and do not show a protruding shape any more, the shape of the endothelial cells is mostly restored to be closely arranged in a short column shape, and the cell length of the endothelial cells is obviously smaller than that of the high-concentration group (P <0.05, figure 1 (b)); at the same time, the peripheral nacrum of dead hemolymph cells in the pearl sac is relatively more deposited.
The research result shows that the medium-concentration LPS injection treatment (0.2 mu g/g) can obviously accelerate the development process of the pearl sac, but the LPS concentration is too high (0.4 mu g/g) to inhibit the development of the pearl sac.
1.5 determination of indicators related to oxidative stress
The superoxide dismutase (SOD) is measured by a Nitro Blue Tetrazolium (NBT) method, and the Catalase (CAT) is measured by an ammonium molybdate colorimetric method. The MDA content is measured by a Thiobarbital (TBA) method.
The results of the level of indicators related to the oxidative stress of the pearl sac at 6 and 12 days after the LPS treatment with different concentrations are shown in FIG. 3, and are as follows: the different Arabic letter designations in the figures differ significantly (P)<0.05). Phenol oxidase (TYR) activity decreased and then increased in LPS-treated groups (0.2 and 0.4. mu.g/g) compared to PBS control group, and there was a significant difference (P) between LPS-treated groups<0.05); pearl sac tissue SOD enzyme activity, MDA and H 2 O 2 The content of the LPS-containing polysaccharide increases along with the increase of the LPS concentration, and the PBS group has a significant difference (P) from the high-concentration group<0.05); CAT enzyme activity increased and then decreased with increasing LPS concentration, but the activity levels of both LPS-treated groups were significantly higher than that of PBS control group (P)<0.05). At 12d after the implantation, the activity level of CAT enzyme activity in the pearl sac tissue is still shown to be changed and reduced after the increase of LPS concentration (P)<0.05), the remaining redox level related indicator levels were not significantly different between the different treatment groups. The results show that LPS treatment mainly affects the oxidation reduction level of the tissues in the early development stage (6d) of the pearl sac, and the oxidation reduction level of the tissues is regulated and controlled by improving the enzyme activity levels of antioxidant enzymes SOD and CAT.
1.6 Gene mRNA expression analysis
Another 3 pearl mussels from each treatment group are separated from the pearl sac and preserved with liquid nitrogen on days 6 and 12 after the operation of the implantation. The oxidative stress-related enzyme activity and the gene mRNA expression level were measured by the fluorescent quantitative PCR method.
The results are shown in FIG. 4, noting: PBS is a control group, LPS50, LPS100 are experimental groups, and the difference in letters in the figure indicates that the different treatment groups have significant difference (P <0.05) at the same time, and indicates that the same treatment groups have significant difference at different times. The relative expression level of LPS-binding receptor protein scavenger receptor SR gene mRNA on cell membranes in pearl sac tissues of pearl mussels of different concentration LPS treatment groups with EF-1 alpha as an internal reference gene is remarkably increased with the increase of LPS concentration 6 days after the implantation (P <0.05), but the relative expression level is reduced when the LPS concentration is increased at 12 days (P < 0.05). Therefore, the research result shows that during the development of the pearl sac, the expression of SR gene mRNA is increased by LPS stimulation treatment activation early development (6 days), but the expression of SR gene mRNA is inhibited by LPS concentration which is excessively high at 12 days of development. The results show that LPS treatment can cause the change of the relative expression level of mRNA of a mode recognition receptor SR gene in a prophenoloxidase system, thereby possibly causing the transmission of extracellular signals of the system into cells and the reaction of downstream phenol oxidase for enzymatically generating melanin and ROS, and the LPS stimulates the activation of the prophenoloxidase system immune pathway in pearl sac tissues of hyriopsis cumingii through the SR gene so as to regulate the oxidation stress level of the pearl sac.
1.7 measurement of amount of deposit of nacre and roundness thereof
Respectively randomly selecting 15-16 pearls and 23-26 pearls to be treated after 42d and 94d of post-grafting operation, dissecting and taking out the pearls, cleaning the pearls with distilled water and drying the pearls for later use. The deposition amount of the pearl is expressed by the weight (mg) of a single pearl of the pearl mussel, the roundness of the pearl is expressed by a value (accurate to 0.01mm) of the diameter of the long axis/the diameter of the short axis of the pearl, and the smaller the value, the better the roundness of the pearl is expressed.
The quality of pearls of pearl-breeding mussels cultured at 42d and 94d after the grafting operation is detected, and the results are shown in the following table 1, wherein the weight of the pearls of the pearl-breeding mussels in the medium-concentration group at 42d after the grafting is obviously greater than that of the pearls in the PBS control group and the high-concentration group (P < 0.05). Although the weight difference of the pearls in each group is not significant at 94d, the roundness of the pearls in the medium concentration group is significantly better than that of the pearls in the PBS control group and the high concentration group (P < 0.05). In combination with the results of the histochemical analysis of the pearl sac, we speculate that LPS treatment affects pearl deposition only in a short time, but LPS treatment at an appropriate concentration (0.2. mu.g/g) can improve the roundness of the pearl over a relatively long time by improving the morphological structure of the pearl sac.
TABLE 1 influence of LPS treatment on epidermal cell length and pearl quality (deposition, roundness) of pearl sac of Unionidae
Note: differential significance P < 0.05; the cell length measurement n is 4, the number n of 42d pearl oysters is 15-16, and the number n of 94d pearl oysters is 23-26.
Example 2 rare earth element injection treatment for regulating oxidative stress level of pearl mussel for promoting pearl sac development and pearl generation
1.1 Experimental materials
The feeding mussels and pearl mussels are 1 year old, healthy and disease-free, and 8-10cm long.
1.2 operation of implanting and breeding pearl mussel
The pearl planting experiment is carried out in the provincial fine breeding field of certain hyriopsis cumingii in Zhejiang in 2018 and 5 months. The hyriopsis cumingii grafting operation is carried out according to a conventional seedless pearl grafting method. All implants were done by a single skilled operator. Culturing the post-operative hyriopsis cumingii with flowing clear water for half a day, transferring to a greenhouse for culturing, and culturing in a hanging net at the water temperature of 27 +/-2 ℃.
1.3 rare earth cerium injection treatment
Implanting into conchospermis after pearl clam, injecting 100 μ l of CeCl 3 The concentration is 0,10 -2 ,10 -3 ,10 -4 ,10 -5 M CeCl 3 10% glucose solution. After 6 days, 3 hyriopsis cumingii pearl sacs were randomly selected from each of 5 treatment groups, fixed with paraformaldehyde solution, and used for histochemical observation.
1.4 Reactive Oxygen Species (ROS) assay
Inner O of blood cells and pearl sac tissue 2 – Horizontal and removal of O 2 – External ROS levels were measured on a flow cytometer (BD Accuri C6) using the Dihydroethidium (DHE) method and the dichlorofluorescein (DCFH-DA) method, respectively, and the active oxygen content was measured as mean fluorescence intensity (Geo mean).
The results are shown in FIG. 7, which shows that the CeCl with proper concentration is injected and treated by the rare earth CeCl3 with different concentrations for 6d 3 (1mM) is capable of reducing ROS levels and superoxide radical levels in blood cells. Therefore, lower ROS and superoxide radical levels contribute to the development of the nacre.
1.5 histochemical analysis
Respectively taking out 2 pearl sacs of hyriopsis cumingii from 5 treatment groups on 6 th and 12 th days after the grafting operation; preparing a tissue sample into a tissue loading piece, embedding, slicing, dewaxing and HE dyeing, and observing the structure of the pearl sac tissue by using a Nikon 80i fluorescence microscope. The 6d results are shown in FIG. 5, and the 12d results are shown in FIG. 6.
6 days after the rare earth cerium injection treatment, the control group (injected with 10% glucose solution) tissue pieces of exocuticle cells were randomly arranged and flattened, CeCl 3 Solutions treatment groups (10) -2 、10 -3 、10 -4 M CeCl 3 ) The exocuticle cells of the tissue slices begin to be columnar and closely arranged, and meanwhile, no granular cells enter the central gaps of the pearl clam tissue slices. The rare earth cerium treatment groups had a higher density of non-granular cells in the central interstitial space of the tissue platelet than the control group and was accompanied by CeCl 3 The treatment concentration increased, the tissue piece center space was larger, and there were more granulocytic free cells. The results show that rare earth cerium promotes the aggregation of blood cells without granular cells and the like in the central gaps of the tissue slices of the pearl mussel so as to accelerate the development and the rapid formation of pearl sacs, and the development degree is closely tangential to the concentration of the rare earth cerium (figure 5, wherein A: 10% G treatment control group (10x 4); B: 10% G treatment control group) -2 M CeCl 3 Treatment group (10x 4); c: 10 -3 M CeCl 3 Treatment group (10x 4); d: 10 -4 M CeCl 3 Treatment group (10x 4); e: 10 -5 M CeCl 3 Treatment group (10x 4); f: local magnification of 10% G treatment group (10x 40); g: 10 -2 M CeCl 3 Treatment group local magnification (10x 40); h: 10 -3 M CeCl 3 Treatment group local magnification (10x 40); i: 10 -4 M CeCl 3 Treatment group local magnification (10x 40); j: 10 -5 M CeCl 3 Treatment group local magnification (10x 40)).
After the rare earth cerium injection treatment for 12 days, the part of the pearl sac cavity in each experimental group is unfolded, and the tissue is in a small piece knotThe connective tissue and the connective tissue of the pearl mussel are obviously fused, the phenomenon that nacre wraps particle-free cells and the particle-free cells gradually die occurs, 10 -3 、10 -4 M CeCl 3 The number of non-granular cells in the pearl sacs of the treated group was less than that of the other treated groups, but 10 -2 M CeCl 3 More particle-free cells were still present in the treated group. The above results show that the appropriate concentration of CeCl 3 Treatment (10) -3 、10 -4 M) promoting the rapid formation of pearl sacs and the deposition of nacres in pearl mussels, high concentration of CeCl 3 Treatment (10) -2 M) has certain inhibiting effect on the development of the pearl sac (figure 6, wherein) A: control group (10x4) treated with 10% G; b: 10 -2 M CeCl 3 Treatment group (10x 4); c: 10 -3 M CeCl 3 Treatment group (10x 4); d: 10 -4 M CeCl 3 Treatment group (10x 4); e: 10 -5 M CeCl 3 Treatment group (10x 4); f: local magnification of 10% G treatment group (10x 40); g: 10 -2 M CeCl 3 Treatment group local magnification (10x 40); h: 10 -3 M CeCl 3 Treatment group local magnification (10x 40); i: 10 -4 M CeCl 3 Treatment group local magnification (10x 40); j: 10 -5 M CeCl 3 Treatment groups were enlarged locally (10x 40).
1.6 determination of indicators related to oxidative stress
The superoxide dismutase (SOD) is measured by a Nitro Blue Tetrazolium (NBT) method, and the Catalase (CAT) is measured by an ammonium molybdate colorimetric method. The MDA content is measured by a Thiobarbital (TBA) method.
The results are shown in FIG. 8 (note: there are significant differences in the different Arabic letter designations in the figure (P)<0.05)), 6 days after the injection treatment, CeCl was compared with the control group 3 SOD (graph A) and CAT (graph B) enzyme activities in the pearl sac tissues of the solution-treated groups were accompanied by CeCl 3 Increase in concentration (P)<0.05), MDA (panel C) concentration with CeCl 3 The increase in concentration is shown as a decrease followed by an increase, 10 -4 The M concentration treatment group was significantly lower than the control group and 10 -2 M concentration treatment group (P)<0.05)。
1.7SEM Crystal Structure analysis
Slicing MargaritaTaking out pearl sac which has developed for 12 days, ultrasonically cleaning with anhydrous alcohol, 75% alcohol, and then Na 2 And immediately taking out the EDTA solution after 8 seconds of treatment, washing with deionized water, and air drying. The crystal structure of the pearl surface was analyzed by using a Japanese Electron (JEOL) JSM-6360LV type scanning electron microscope.
The results are shown in FIG. 9 (A: 10% glucose solution control group (5000X); B: 10) -2 M CeCl 3 Treatment (5000 ×); c: 10 -3 M CeCl 3 Treatment (5000 ×); d: 10 -4 M CeCl 3 Treatment (5000 ×); e: 10 -5 M CeCl 3 Treatment (5000X)) for different concentrations of rare earth CeCl 3 The observation of a scanning electron microscope of pearls treated for 12 days by injection shows that most of the crystals in the pearl layer of the control group are in an amorphous ACC phase, and part of the crystals are in a spherical poly-aggregation state and are aragonite. 10 -3 And 10 -4 M CeCl 3 The crystal morphology of the treated group is predominantly hexagonal with gaps between them. 10 -2 M CeCl 3 In addition to regular hexagonal crystals, some organic matter was present in the treatment group (indicated by arrows in the figure). 10 -5 M CeCl 3 The crystal morphology of the treated group is close to hexagonal, and is more regular compared with the crystal morphology of the control group, and larger gaps exist among the crystals. The above results indicate that the appropriate concentration of CeCl 3 Treatment (10) -3 、10 -4 M) promoting the improvement of the surface crystal structure (aragonite structure) of nacre of the pearl mussel.
1.8 measurement of the amount of sediment in the nacre and the roundness thereof
On day 12, 2 and 4 months after the grafting operation, 4, 11 and 24 hyriopsis cumingii are randomly selected from 5 treatment groups respectively, pearls are taken out, soaked in 5% NaOCl solution for 10min, washed clean by distilled water, and dried to determine the deposition amount. Wherein the pearl deposition amount of 2-4 months is represented by average single-particle pearl mass (mg), and the roundness is represented by major axis diameter/minor axis diameter.
The results are shown in Table 2, with different concentrations of CeCl 3 After 2 months of solution treatment, 10 months after solution treatment, compared with the control group -3 M CeCl 3 The quality of single-grain pearls in the concentration treatment group is higher than that of other groups, butThe difference was not significant; CeCl 3 The roundness of the pearl in the concentration treatment group is obviously better than that in the control group, and the pearl roundness is along with CeCl 3 The better the roundness of the pearl is (P is less than 0.05) when the concentration is increased; after 9 months of treatment, the quality of a single pearl is 10 -4 M CeCl 3 The concentration treatment group was significantly higher than the control group and 10 -2 M CeCl 3 A concentration treatment group; roundness of pearl 10 -3 M CeCl 3 Concentration treatment and 10 -4 M CeCl 3 The concentration treatment was significantly higher than the control group and 10 -2 M CeCl 3 Concentration treatment group. The results show that the rare earth cerium can promote early-stage pearl sac development of hyriopsis cumingii and improve pearl deposition and roundness.
TABLE 2 comparison of the deposition amount and roundness of the nacre of hyriopsis cumingii treated by rare earth cerium with different concentrations at 2 and 4 months
Note: the roundness is expressed by the major axis diameter/minor axis diameter of the pearl, and the value is as good as the roundness approaches 1. The different arabic letters in the table indicate significant differences (P < 0.05).
Therefore, the rare earth cerium treatment with an appropriate concentration can promote the development of the pearl sac of hyriopsis cumingii at the early stage and the growth of the pearl by increasing the activity of antioxidant enzymes of tissues such as the pearl sac of the hyriopsis cumingii and reducing the level of oxidative stress.
Example 3 adding different amounts of taurine into aquaculture water to promote the sediment amount of nacre of pearl mussel
1.1 Experimental materials and methods
For experiments, 1-year hyriopsis cumingii is purchased from a freshwater pearl culture base in Jinhuawuyi county, and the insertion is firstly carried out according to a conventional method, and each hyriopsis cumingii is inserted with 28 small cell pieces. The length of the test hyriopsis cumingii is 7.2-8.3cm, and the weight is 32-38 g.
1.2 cultivation and treatment of pearl mussels
In the experiment, a plurality of 200L aquariums are selected to simulate outdoor microflow water body for indoor culture. 12 hyriopsis cumingii are placed in each water tank, and the treatment concentration is repeated for 2 times. The basic bait is prepared by mixing chlorella concentrated solution (3g/L), fresh soybean milk concentrated solution (7.5g/L) and dry yeast (10mg/ml), and specifically adding 1g of dry yeast and 2ml of fresh soybean milk concentrated solution into 100ml of chlorella concentrated solution before feeding. The treatment group fed with the basic bait was used as a control group, the treatment group added with 1% by mass of taurine in the fed basic bait was used as a low-concentration taurine addition group, and the treatment group added with 2% by mass of taurine in the fed basic bait was used as a high-concentration taurine addition group. During the experiment, the treatment groups are fed with baits for 3 times every morning, noon and evening, the water bodies of the groups are oxygenated regularly, the dissolved oxygen is controlled to be 6.5 +/-0.2 mg/L, the temperature is controlled to be 26 +/-1 ℃, and the pH value is 6.8 +/-0.2. The treatment solutions were changed every 10 days. After the pearl mussels are cultured in the room for 30d and 60d respectively, the liver and pancreas tissues of the hyriopsis cumingii are dissected and taken from each water tank at low temperature and are stored at 20 ℃.
1.3 determination of indicators related to oxidative stress
The superoxide dismutase (SOD) is measured by a Nitro Blue Tetrazolium (NBT) method, and the Catalase (CAT) is measured by an ammonium molybdate colorimetric method. The MDA content is measured by a Thiobarbital (TBA) method.
As shown in FIG. 10 (in the figure, significant difference (P <0.05) is shown by different Arabic letters), the enzyme activity levels of SOD and CAT in the liver and pancreas tissues of the group with low concentration of taurine are significantly higher than that of SOD enzyme activity level (P <0.05) of the control group when the pearl mussel is cultured for 30d by taurine treatment. The enzymatic activities of SOD and CAT in the liver and pancreas tissues of the taurine high-concentration added group were inhibited at 60d, and were lower than those of the control group and the low-concentration added group (P <0.05), but the enzymatic activity level in the low-concentration added group was still significantly higher than that of the control group (a, b of fig. 10). The results show that the addition of a proper amount of taurine in the aquaculture water body can improve the activities of antioxidase SOD and CAT enzymes of liver and pancreas tissues so as to be beneficial to reducing the oxidative stress level after the pearl mussel graft operation, but the addition amount of taurine cannot be increased or even can possibly inhibit the activity of SOD enzymes after long-term (60d) addition.
When the pearly mussels are cultured for 30d and 60d, the level of Malondialdehyde (MDA) which is a lipid peroxidation product of the low-concentration taurine-added group of liver and pancreas pearly mussels is slightly lower than that of a control group (the MDA is averagely reduced by 8.7% when the pearly mussels are cultured for 30d, and is averagely reduced by 3.3% (P <0.05) when the pearly mussels are cultured for 60d), and no significant difference exists.
1.4 determination of amount of deposited nacre
Taking 10 pieces of the hyriopsis cumingii treated by each concentration, repeatedly washing the hyriopsis cumingii with tap water, wiping the hyriopsis cumingii dry, taking out each hyriopsis cumingii pearl, wiping the hyriopsis cumingii pearl dry with absorbent paper, weighing, recording the weight of each hyriopsis cumingii pearl, and calculating the sediment amount of nacre by the average weight of each pearl in mg.
The results are shown in (d) of fig. 10, when the pearls of the pearl mussels cultured by adding low-concentration taurine are cultured for 30d and 60d, the deposition amount of the pearls of the pearl mussels cultured by adding low-concentration taurine is obviously higher than that of the control group (the average deposition amount is increased by 27.5% when the pearls are cultured for 30d, and the average deposition amount is 51.8% (P <0.05) when the pearls are cultured for 60 d).
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (8)
1. A method of regulating the production of the nacre sac of a pearl mussel, which comprises reducing the level of oxidative stress of the pearl mussel after a nucleus or a sheet grafting operation;
reducing the level of oxidative stress by inhibiting the pro-phenoloxidase system ROS production pathway;
a method for inhibiting the ROS production pathway of the prophenoloxidase system comprises activating the antioxidant enzyme system of pearl mussel or inhibiting the enzymatic activity of phenoloxidase by a phenoloxidase inhibitor using a pathogen pattern recognition molecule;
pathogen pattern recognition molecules include lipopolysaccharides, peptidoglycans, lipoteichoic acids, mannans, glucans, or mannose.
2. The method of claim 1, wherein the pathogen pattern recognition molecule is a lipopolysaccharide.
3. The method of claim 1, wherein the phenol oxidizing enzyme inhibitor comprises 1-phenyl-2-thiourea, 4-hexylresorcinol, or kojic acid.
4. The method of claim 1, wherein the lipopolysaccharide activation method comprises: injecting lipopolysaccharide at the muscle of adductor muscle according to the dose of 0.1-0.3 mug/g mussel weight within 0-6 days after nucleus planting or sheet planting operation of the pearl mussel.
5. The method according to any one of claims 1 to 4, wherein the temperature of the water body for cultivating pearl mussel is 25-29 ℃.
6. The method of claim 5, wherein the pearl mussel is a hyriopsis cumingii.
7. The method of claim 6, wherein the pearl mussel is a 1-year-old hyriopsis cumingii with a shell length of 8-10 cm.
8. A method for producing pearls by pearl culturing mussels, which is characterized in that the pearl sacs are cultured by the method of any one of claims 1 to 7, and the pearl sacs containing the pearl sacs are cultured to obtain pearls.
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