Vertical section-based naked carp otolith embedding method and otolith grinding sheet preparation method and application

Abstract

The invention belongs to the technical field of fish ecology, and particularly relates to a nude carp otolith embedding method based on a longitudinal section and a preparation method and application of an otolith grinding sheet. The epoxy resin and the curing agent are mixed according to a certain mass ratio and then placed on the vegetable parchment to embed the longitudinal section of the naked carp otolith, the vegetable parchment is resistant to high temperature and high in strength and is not prone to deformation caused by high temperature, and the cured embedding piece is easily separated from the vegetable parchment; the embedding agent is low in shrinkage rate after being cured, the otolith is not prone to fracture, the hardness requirement of subsequent grinding can be met, adhesion is high, degumming is avoided in the subsequent grinding process, and fragmentation of the naked carp otolith is effectively avoided. According to the method, the embedding piece polished for the first time is immediately immersed in water, oxidation of the polished surface of the otolith can be prevented, subsequent polishing is continued, and the otolith grinding piece with clear wheel lines is obtained. The method is simple in operation process, does not need to use a high-cost and low-efficiency embedding mold, reduces the preparation cost, and improves the preparation efficiency.

Description

Technical Field

[0001] This invention belongs to the field of fish ecology technology, specifically relating to a method for embedding otoliths in longitudinal sections of naked carp and a method for preparing otolith grinding discs and their applications. Background Technology

[0002] Fish, being lower vertebrates, cannot remember and record their life history processes such as age, habitat characteristics, and migration patterns like humans. Therefore, they typically rely on calcified tissues such as scales, vertebrae, operculum, and otoliths to reconstruct their life history. Otoliths are deposits located in the inner ear of fish, composed of calcium carbonate, small amounts of organic matter, and trace elements. They include three pairs of otoliths: microotoliths, asterotoliths, and sagittalotoliths, distributed in the utricle, saccule, and saccule, respectively, and are primarily responsible for hearing and balance in fish. Compared to other calcified tissues, otoliths are characterized by metabolic inertia and continuous growth, maintaining positive growth even in older fish. The microstructures formed and the trace elements deposited can completely record the entire life history of fish. Currently, otoliths have become an important tool in fish ecology research, including age determination, habitat reconstruction, growth regression, and aquatic environment remodeling, and have given rise to theories and techniques such as otolith microchemistry and hard tissue chronology. When conducting the aforementioned research based on otoliths, it is necessary to prepare otolith grinding discs to obtain smooth and clear annual or daily ring structures for subsequent measurement and analysis. Typically, the clarity of the ring patterns on the sagittal section of the otolith is sufficient for the research requirements. Furthermore, the sagittal section has a relatively large cross-sectional area and relatively short inner and outer axes, requiring only five steps to prepare an otolith grinding disc: otolith removal, cleaning, fixing, grinding, and sealing. However, the clarity of the ring patterns on the three sections of the otolith varies among different fish species, and not all fish otoliths have sufficient clarity on the sagittal section for the research. In such cases, otolith grinding discs need to be prepared based on longitudinal or transverse sections. However, because the cross-sectional areas of both the longitudinal and transverse sections of the otolith are relatively small, and the dorsal and ventral axes and cephalotail axes are relatively long, conventional otolith fixing methods cannot meet the requirements for stability during grinding. Chinese patent CN202411210238.7 adds otolith embedding and otolith cutting steps. By cutting the otoliths to reduce the length of the dorsal or ventral axis or head-to-tail axis, the requirements for otolith fixation are met. However, this method has drawbacks such as cumbersome process, high cost, and low efficiency. Chinese patent CN201310147893.8 uses hot melt adhesive to fix the otoliths. Although it can meet the requirements for firmness during otolith grinding and has the advantages of simple process, low cost, and high efficiency, the hardness of the hot melt adhesive after curing is insufficient and its adhesion is weak. It is extremely difficult to grind on sandpaper. If forced to grind, it is easy to cause the otoliths to delaminate or break, ultimately resulting in poor otolith grinding disc preparation. Summary of the Invention

[0003] The purpose of this invention is to provide mass-produced otolith grinding discs for naked carp, solving the problems of complex operation, high cost, and low efficiency when using the longitudinal section as the otolith grinding surface, and making the resulting otolith grinding discs for naked carp have a clear wheel pattern structure.

[0004] This invention provides a method for embedding otoliths in naked carp, comprising the following steps: Epoxy resin and curing agent are mixed at a mass ratio of (4~6):1. Air bubbles in the mixture are removed to obtain an embedding agent. The embedding agent is placed on sulfuric acid paper, and then the longitudinal section of the otolith of a naked carp is inserted into the embedding agent with the longitudinal section as the adhesion surface. The mixture is then dried until fully cured.

[0005] Preferably, the volume ratio of the embedding agent to the otoliths of naked carp is 2-4 mL: 1 otolith.

[0006] Preferably, the drying method includes air drying at a temperature of 5~30°C.

[0007] Preferably, the sulfuric acid paper is divided into multiple regions, with dividing lines between adjacent regions, and the embedding agent and the otoliths of naked carp are placed in each region.

[0008] Preferably, each region is a 5cm × 5cm square.

[0009] Preferably, the otoliths of the naked carp are microotoliths of the naked carp; the curing agent includes fatty amine adducts.

[0010] This invention provides the application of the embedding method described above in the preparation of otoliths from naked carp.

[0011] This invention provides a method for preparing otoliths from naked carp, comprising the following steps: The otoliths of naked carp were embedded according to the embedding method described in the above technical solution; Remove the epoxy resin sheet containing the otoliths of the naked carp from the sulfuric acid paper, and polish it with 400#~800# wet sandpaper until the edge ring structure of the naked carp otoliths is vaguely visible and the core area ring structure is not visible. Immediately immerse it in water to obtain the first polished product. The polished surface of the first polished product is placed face down on a glass slide coated with nail polish. After covering with nail polish and drying, the fixed otoliths of naked carp are obtained. Use 400#~800# wet sandpaper to coarsely polish the fixed otoliths of the naked carp. When the core area is visible, switch to 1000#~1500# wet sandpaper for fine polishing. Polish until the otolith core is exposed and the wheel pattern is clearly visible. Then polish and seal to obtain the otolith grinding disc of the naked carp.

[0012] Preferably, the drying time after applying the nail polish is 4-6 hours, and the drying method is air drying.

[0013] The present invention provides that the otoliths of naked carp prepared by the preparation method described above can be used in the ecological research of naked carp.

[0014] Beneficial effects: This invention involves embedding the otoliths of naked carp into longitudinal sections by mixing epoxy resin and a curing agent in a specific mass ratio. The mixture cures at room temperature, simplifying the process. It is pollution-free, harmless to the environment and laboratory personnel, exhibits low shrinkage after curing, and minimizes the risk of breakage in the otoliths, ensuring the required hardness for subsequent polishing. Furthermore, it demonstrates strong adhesion, preventing delamination during polishing and effectively avoiding otolith fragmentation. The invention also involves pouring the heated embedding agent onto tracing paper. Tracing paper is heat-resistant and strong, preventing deformation due to high temperatures. While the embedded pieces adhere to the paper after curing, the high strength of the tracing paper allows for easy removal, eliminating the need for costly molds and significantly improving the efficiency of the embedding process.

[0015] Furthermore, this invention enables the simultaneous embedding of multiple otoliths from naked carp by marking lines on sulfuric acid paper and dividing the area into regions, thereby achieving the batch preparation of otolith grinding discs from naked carp and improving the efficiency of otolith grinding disc preparation.

[0016] The present invention embeds otoliths of naked carp according to the embedding method described above. The otoliths of naked carp are immediately immersed in water after the first polishing, which can prevent oxidation of the polished surface and effectively solve the problem of unclear wheel pattern structure caused by oxidation of the polished surface. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 Flowchart for preparing otoliths from naked carp; Figure 2 Epoxy resin sheet used in the otolith embedding process; Figure 3 The wheel-like texture is formed by oxidation of the polished surface of the otolith. Detailed Implementation

[0019] This invention provides a method for embedding otoliths in naked carp, comprising the following steps: Epoxy resin and curing agent are mixed at a mass ratio of (4~6):1. Air bubbles in the mixture are removed to obtain an embedding agent. The embedding agent is placed on sulfuric acid paper, and then the longitudinal section of the otolith of a naked carp is inserted into the embedding agent with the longitudinal section as the adhesion surface. The mixture is then dried until fully cured.

[0020] In this invention, epoxy resin and curing agent are mixed at a mass ratio of (4~6):1, and air bubbles in the mixture are removed to obtain an embedding agent.

[0021] Because the otoliths of naked carp are very small, they need to be embedded and fixed to facilitate subsequent fine polishing. Therefore, the selection of the embedding agent for naked carp otoliths is particularly important. This invention utilizes epoxy resin, which has the following advantages: (1) it can be cured at room temperature to simplify the operation process; (2) it has a low shrinkage rate after curing, making it less likely for naked carp otoliths to break; (3) it has strong adhesion to ensure that it does not delaminate during polishing, effectively avoiding the breakage of naked carp otoliths; and (4) it is pollution-free and does not harm the environment or experimental personnel. If epoxy resin is replaced with other embedding agents such as paraffin or acrylic resin, it is impossible to simultaneously meet the requirements of low shrinkage rate, strong adhesion, and pollution-free, ultimately affecting the embedding effect of naked carp otoliths and thus affecting the preparation effect of subsequent naked carp otolith grinding discs.

[0022] In one embodiment, the mass ratio of epoxy resin to curing agent in this invention is 5:1. In another embodiment, the curing agent includes aliphatic amine adducts. Since the otoliths of naked carp require suitable hardness after embedding to meet the requirements of subsequent polishing, if the hardness is too low, the otoliths are prone to debonding or breakage during polishing; if the hardness is too high, it will increase the time cost of the polishing process. This invention limits the mass ratio of epoxy resin to curing agent to ensure the hardness requirements for subsequent polishing; if other ratios are used, suitable hardness cannot be obtained.

[0023] In one embodiment, the method for removing air bubbles from the mixture according to the present invention includes drying. In one embodiment, the drying method includes baking at a temperature of 55-65°C; in another embodiment, the baking temperature is 60°C. In one embodiment, the drying time is 5-10 minutes; in another embodiment, the baking time is 8 minutes.

[0024] After obtaining the embedding agent, the present invention places the embedding agent on sulfuric acid paper, and then inserts the longitudinal section of the otolith of a naked carp into the embedding agent, and dries it until it is completely cured.

[0025] This invention uses the longitudinal section of the otoliths of naked carp as the attachment surface. Compared with the sagittal and transverse sections of the otoliths of naked carp, it is easier to prepare otolith grinding discs with clear concentric rings for use in the ecological research of naked carp.

[0026] In one embodiment, the volume ratio of the embedding agent to the otoliths of *Gymnocypris chinensis* is 2-4 mL: 1 otolith; in another embodiment, the volume ratio of the embedding agent to the otoliths of *Gymnocypris chinensis* is 3 mL: 1 otolith. In one embodiment, the otoliths of *Gymnocypris chinensis* are microotoliths of *Gymnocypris chinensis*.

[0027] In one embodiment, the drying method of the present invention includes air drying. In one embodiment, the air drying time is 2-4 hours; in another embodiment, the air drying time is 3 hours. In one embodiment, the drying temperature is 5-30°C; in another embodiment, the drying temperature is 15-25°C. In one embodiment, the present invention divides the sulfuric acid paper into multiple regions, wherein there are dividing lines between adjacent regions, and the embedding agent and the otoliths of naked carp are placed in each region. In another embodiment, each region of the present invention is a 5cm × 5cm square.

[0028] Traditional embedding methods require pouring the embedding agent into a mold. Although a release agent is applied to the inner surface of the mold before pouring, removing the embedded block from the mold after the embedding agent has solidified is still difficult, leading to low embedding efficiency. This invention pours the embedding agent onto the surface of tracing paper. After solidification, the resulting embedded disc can be easily removed from the tracing paper, significantly improving the efficiency of the embedding process. Furthermore, otolith grinding discs typically require batch production. Traditional embedding methods necessitate the purchase of large quantities of costly molds. The tracing paper used in this invention is very inexpensive, and by marking lines on the tracing paper to divide the area, multiple otoliths from *Gymnocypris* can be embedded simultaneously, enabling batch production of *Gymnocypris* otolith grinding discs. This improves the efficiency of otolith grinding disc production while reducing costs.

[0029] Furthermore, the use of sulfuric acid paper in the embedding process of this invention has the following advantages: (1) it is easy to write on and the written content will not be erased due to accidental touch during the experimental operation; (2) it is heat resistant and will not deform due to high temperature; (3) it has high strength. After the embedding agent is cured, it will stick to the paper. If the paper is not strong enough, it is very easy to stick to the paper when the cured embedding sheet is peeled off, which will cause inconvenience to the subsequent polishing process. If the sulfuric acid paper of this invention is replaced with plastic paper, ordinary paper, kraft paper, etc., the above characteristics cannot be met at the same time, which will ultimately affect the embedding effect of otoliths in naked carp, and thus affect the preparation effect of otolith grinding sheets of naked carp.

[0030] Given the advantages of the embedding method provided by this invention, the application of the embedding method described above in the preparation of otoliths from naked carp is also within the scope of protection of this invention.

[0031] This invention provides a method for preparing otoliths from naked carp, comprising the following steps: The otoliths of naked carp were embedded according to the embedding method described in the above technical solution; Remove the epoxy resin sheet containing the otoliths of naked carp from the sulfuric acid paper, and polish it with 400#~800# wet sandpaper until the edge ring structure of the otoliths of naked carp is vaguely visible and the core area ring structure is not visible. Immediately immerse it in water to obtain the first polished product. The polished surface of the first polished product is placed face down on a glass slide coated with nail polish. After covering with nail polish and drying, the fixed otoliths of naked carp are obtained. Use 400#~800# wet sandpaper to polish the fixed otoliths of the naked carp. When the core area is visible, switch to 1000#~1500# wet sandpaper for fine polishing. Polish until the otolith core is exposed and the wheel pattern is clearly visible. Then polish and seal to obtain the otolith polishing disc of the naked carp.

[0032] In one embodiment, the drying time after applying nail polish is 4-6 hours; in another embodiment, the drying time is 5 hours. In one embodiment, the drying method is air drying. The nail polish has high transparency, and this invention utilizes the fact that the nail polish application does not affect the observation of the otolith microstructure; furthermore, nail polish is readily available on the market, requires no special preparation, is inexpensive, and has a suitable hardness after curing, meeting the requirements for durability.

[0033] In one embodiment, the polishing process of the present invention utilizes polishing paper. In another embodiment, the polishing paper of the present invention is made of aluminum oxide. In yet another embodiment, the sealing process of the present invention utilizes nail polish.

[0034] This invention first uses 400#~800# wet sandpaper to sand the first surface of the otolith of the naked carp until the edge concentric rings are vaguely visible, while the core area concentric rings are not visible. After flipping it over, the fixed otolith of the naked carp is then coarsely sanded using 400#~800# wet sandpaper until the core area is visible. Then, 1000#~1500# wet sandpaper is used for fine sanding. This effectively avoids the problem of blurred concentric rings caused by over-sanding. Furthermore, this invention immediately immerses the otolith of the naked carp in water after the first sanding to prevent oxidation of the sanded surface, effectively solving the problem of unclear concentric rings caused by oxidation of the sanded surface.

[0035] This invention provides the application of the otoliths of naked carp prepared by the preparation method described above in the ecological research of naked carp.

[0036] As one implementation method, the ecological study of naked carp described in this invention includes one or more of the following: age identification of naked carp, habitat characteristics, and migration patterns.

[0037] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for embedding otoliths in longitudinal sections of naked carp, a method for preparing otolith grinding discs, and their applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1 according to Figure 1 The specific steps for preparing otoliths from naked carp are as follows: 1. Otolith sampling: After anesthetizing the naked carp with MS-222 (ethyl m-aminobenzoate methanesulfonate), a pair of microotoliths were removed from the utricle of the inner ear of the naked carp using dissecting scissors and forceps; 2. Otolith cleaning: Clean the dirt on the micro-otoliths with ethanol, air dry, and store in 0.5 mL centrifuge tubes; 3. Otolith embedding: Draw a grid of approximately 5 cm squares on tracing paper using a black marker, and label the lower right corner of each grid with the corresponding otolith number. Mix epoxy resin and curing agent (fatty amine adduct) in a disposable paper cup at a mass ratio of 4-6:1. After thorough mixing, place the mixture in a 60°C oven for 5-10 minutes to remove air bubbles. While still hot, pour approximately 3 mL of the mixture into each grid. Immediately using tweezers, insert the micro-otoliths into the mixture, with the longitudinal section as the adhesion surface. Allow to air dry for 2-4 hours until fully cured. Figure 2 ); 4. Otolith polishing: Remove the hardened epoxy resin sheet from the tracing paper. Use 400#~800# wet sandpaper to polish the embedded microotoliths. Observe under an optical microscope every 10~20 seconds until the edge concentric rings of the microotoliths are vaguely visible and the core area concentric rings are no longer visible. Immediately after polishing, immerse the microotoliths in water to prevent oxidation of the polished surface, which would affect the clarity of the microotolith concentric rings. Figure 3 ); 5. Secondary polishing: Cut the epoxy resin sheet to the appropriate size using scissors. Apply a layer of clear nail polish to the slide using a small brush. Immediately place the polished side of the micro otoliths face down on the nail polished area of ​​the slide. Then, drop 1-2 drops of clear nail polish onto the epoxy resin sheet and let it air dry for 4-6 hours. Use 400#-800# wet sandpaper to coarsely polish the fixed micro otoliths. When the core area is visible, switch to 1000#-1500# wet sandpaper for fine polishing. Observe under an optical microscope every 5-10 seconds of polishing until the otolith core is exposed and the concentric rings are clearly visible. 6. Otolith polishing: Polish the polished otolith discs with polishing paper (alumina), and then rinse the otoliths with distilled water for 10 seconds. 7. Otolith sealing: Immediately after cleaning the otolith grinding disc, use a small brush to lightly apply a layer of clear nail polish to the surface of the disc to prevent oxidation and dust from interfering with observation. After the nail polish dries, it can be stored for subsequent measurement and analysis.

[0039] As can be seen from the above, the present invention can use the longitudinal section of the otolith of naked carp as the polishing surface to efficiently prepare otolith grinding discs of naked carp. The operation is simple, the cost is low, and the resulting otolith grinding discs of naked carp have clear wheel texture structure.

[0040] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for embedding otoliths in naked carp, characterized in that, Includes the following steps: Epoxy resin and curing agent are mixed at a mass ratio of (4~6):

1. Air bubbles in the mixture are removed to obtain an embedding agent. The embedding agent is placed on sulfuric acid paper, and then the longitudinal section of the otolith of a naked carp is inserted into the embedding agent with the longitudinal section as the adhesion surface. The mixture is then dried until fully cured.

2. The embedding method according to claim 1, characterized in that, The volume ratio of the embedding agent to the otoliths of naked carp is 2-4 mL: 1 otolith.

3. The embedding method according to claim 1, characterized in that, The drying method includes air drying at a temperature of 5~30℃.

4. The embedding method according to claim 1, characterized in that, The sulfuric acid paper is divided into multiple regions, with dividing lines between adjacent regions. The embedding agent and the otoliths of naked carp are placed in each region.

5. The embedding method according to claim 4, characterized in that, Each region is a 5cm x 5cm square.

6. The embedding method according to any one of claims 1 to 5, characterized in that, The otoliths of the naked carp are microotoliths of the naked carp; the curing agent includes fatty amine adducts.

7. The application of the embedding method according to any one of claims 1 to 6 in the preparation of otoliths from naked carp.

8. A method for preparing otoliths from naked carp, characterized in that, Includes the following steps: The otoliths of naked carp are embedded according to any one of claims 1 to 7; Remove the epoxy resin sheet containing the otoliths of naked carp from the sulfuric acid paper, and polish it with 400#~800# wet sandpaper until the edge ring structure of the otoliths of naked carp is vaguely visible and the core area ring structure is not visible. Immediately immerse it in water to obtain the first polished product. The polished surface of the first polished product is placed face down on a glass slide coated with nail polish. After covering with nail polish and drying, the fixed otoliths of naked carp are obtained. Use 400#~800# wet sandpaper to coarsely polish the fixed otoliths of the naked carp. When the core area is visible, switch to 1000#~1500# wet sandpaper for fine polishing. Polish until the otolith core is exposed and the wheel pattern is clearly visible. Then polish and seal to obtain the otolith grinding disc of the naked carp.

9. The preparation method according to claim 8, characterized in that, The drying time after applying the nail polish is 4-6 hours, and the drying method is air drying.

10. The application of the otoliths of naked carp prepared by the preparation method according to claim 8 or 9 in the ecological research of naked carp.

Images