Improved euchiloglanis kishinouyei chromosome preparation method

Abstract

The invention provides an improved euchiloglanis kishinouyei chromosome preparation method, which comprises the following steps: gradually raising the temperature and injecting PHA twice: in the euchiloglanis kishinouyei temporary culture process, raising the water temperature from 18 DEG C to 20 DEG C within 12 hours, injecting a first needle of PHA, raising the temperature to 22.5-23 DEG C after 12 hours, injecting a second needle of PHA, and keeping the water temperature at 22.5-23 DEG C unchanged; after the PHA is injected for the second time for 3 hours, colchicine is injected; sampling: washing the head and kidney in normal saline, and collecting cells; 0.5% of KCl is used for hypotonic treatment; fixing with a Carnoy's fixing solution, and centrifuging; dropping by a cold dropping method and naturally drying; and carrying out Giemsa dyeing and flaking. According to the method, the euchiloglanis kishinouyei somatic cell proliferation is promoted through heating and twice PHA injection, so that the metaphase division phase is increased, the form is clear and dispersed, and the karyotype quality and the preparation success rate of the chromosome are remarkably improved.

Description

Technical Field

[0001] This invention relates to a method for preparing fish chromosomes, specifically to an improved method for preparing chromosomes of the Huangshi catfish. Background Technology

[0002] The Huangshi catfish, also known as the stone catfish or Huangshi catfish, belongs to the order Siluriformes, family Sociidae, and genus Sociidae. This species is a small, benthic fish endemic to the upper reaches of the Yangtze River, mainly distributed in the main streams and tributaries of the Jinsha and Minjiang Rivers, covering Qinghai, Sichuan, Yunnan, and Tibet. Its typical habitat is mountain streams, creeks, and fast-flowing waters in small rivers in remote mountainous areas. The Huangshi catfish is a cold-water fish, with an optimal survival temperature of 14-18℃. Due to its tender and delicious flesh, the Huangshi catfish is considered a valuable economic fish in its production areas, commanding a very high market price. However, due to overfishing and habitat changes, its natural population is now scarce and difficult to catch.

[0003] Chromosomes, as the primary carriers of genetic information, are crucial for cytogenetics research, and their number and karyotype analysis are central to understanding species evolution, taxonomic relationships, and fish genetics and breeding. Chromosome preparation is fundamental to karyotype analysis, requiring the acquisition of metaphase chromosomes with high mitotic indices and clear morphology. Differences in tissue structure, physiological state, and processing conditions among different fish species result in variations in the quantity and quality of mitotic phases. Therefore, establishing a stable and efficient species-specific chromosome preparation system requires systematic optimization and repeated validation to provide a reliable cytological basis for subsequent research.

[0004] Currently, the number of crocodile species for which karyotype studies have been conducted is very limited, mainly including black-spotted crocodile, yellow-stone crocodile, green-stone crocodile, Fujian striped-breasted crocodile, and Chinese striped-breasted crocodile (Li Shushen et al. 1981, Yu Xianjue et al. 1989, Ren Xiuhai 1992). A review of existing literature reveals that the preparation of chromosomes for scad and most other fish species generally employs the "PHA in vivo injection-colchicine treatment" method (Wu Yunfei 1999, Zhu Chuankun and Pan Zhengjun 2022). This method can be summarized as follows: 1. PHA injection: a single intrathoracic or intraperitoneal injection at a dose of 5-15 μg / g of fish body weight; 2. Colchicine treatment: approximately 2-24 hours after PHA injection, 1-6 μg / g of colchicine is injected and treated for 2-6 hours to accumulate metaphase; 3. Sampling and slide preparation: usually, head kidney or kidney tissue is taken, and chromosome specimens are prepared through steps such as hypotonic treatment, fixation, slide preparation, and Giemsa staining. Although the above-mentioned conventional methods have been successful in many fish species, they face significant challenges when applied to scad, especially plateau-endemic species like the yellowstone scad, and it is difficult to obtain ideal results: Zhang Lin (2023) pointed out in summarizing the experience of seven difficult-to-treat aquatic animals that different groups have different sensitivities and response patterns to PHA.

[0005] Experiments using existing traditional chromosome karyotype preparation methods yielded a small number of metaphase chromosomes from the Huangshi catfish, which were unevenly distributed and had unclear morphology. Figure 1 As shown. Therefore, there is an urgent need for a stable and reliable chromosome preparation method optimized for the biological characteristics of the Huangshi catfish. Summary of the Invention

[0006] This invention addresses the problems of low number, uneven dispersion, and unclear morphology of metaphase chromosomes in cold-water fish such as the Huangshi catfish, by providing an improved method for preparing Huangshi catfish chromosomes. Based on extensive reference to classical methods, this invention systematically optimizes and innovates key steps. We employ a method of gradual temperature increase followed by two injections of PHA, effectively promoting somatic cell proliferation in Huangshi catfish, resulting in a larger number of metaphase chromosomes with clear morphology, good dispersion, and complete number. This improved preparation method effectively enhances the quality and success rate of chromosome karyotype preparation.

[0007] This invention provides an improved method for preparing chromosomes of the Huangshi catfish, which is carried out according to the following steps: 1. During the temporary rearing of Huangshi mantis shrimp, the water temperature is raised from 18℃ to 20℃ within 12 hours, and the mantis shrimp are injected with the first dose of PHA; after 12 hours, the water temperature is raised to 22.5-23℃, and the mantis shrimp are injected with the second dose of PHA, while maintaining the water temperature at 22.5-23℃; after 3 hours, colchicine solution is injected. 2. After 3 hours, cut off the gills and tail of the Huangshi catfish and bleed it in water. Dissect the fish, remove the head and kidney, place them in a petri dish containing freshwater fish saline to clean them, remove blood clots, and place them in a culture dish containing physiological saline. Use tweezers to grind the head and kidney tissue, aspirate it into a 10mL centrifuge tube and repeatedly blow it with a pipette, add physiological saline, mix well, centrifuge at 1200r / min for 12min, and discard the supernatant. 3. Add 5 mL of 0.5% KCl hypotonic solution to the product of step 2, mix well, let stand at room temperature for 30 min for hypotonic treatment, add 1 mL of Carnoy fixative prepared in the ratio of methanol: glacial acetic acid = 3:1, mix well, centrifuge at 1200 r / min for 12 min, discard the supernatant, and mix well. 4. Add 5 ml of Carnoy's fixative to the product from step 3. Use a dropper to mix the product and Carnoy's fixative evenly. Let it stand at room temperature for 20 min, then centrifuge at 1200 r / min for 10 min and discard the supernatant. 5. Repeat step four once, then discard the supernatant; 6. Add 5 ml of Carnoy's fixative to the product from step 5, mix well by pipetting, let stand at room temperature for 10 min, then centrifuge at 1200 r / min for 10 min and discard the supernatant. 7. Add 0.5 ml of Carnoy's fixative to the cell pellet after centrifugation in step 6, mix well to prepare a cell suspension, draw the cell suspension into a dropper, drop it onto a pre-frozen glass slide, and then pass the glass slide over an alcohol lamp flame 2-3 times and let it air dry at room temperature. 8. Stain the dried chromosome slide from step 7 with 10% Giemsa stain for 10 minutes, rinse with tap water, and air dry to complete chromosome specimen preparation, thus obtaining the Huangshi catfish chromosome.

[0008] Furthermore, in step one, when temporarily holding the Huangshi catfish, the water temperature is raised from 18℃ to 20℃ within 12 hours. The Huangshi catfish is injected with the first dose of PHA at a ratio of 6μg:1g of PHA to fish body weight. After 12 hours, the water temperature is raised to 22.5-23℃, and the Huangshi catfish is injected with the second dose of PHA at a ratio of 6-8μg:1g of PHA to fish body weight. The water temperature is maintained at 22.5-23℃.

[0009] Furthermore, in step one, 3 hours after the second injection of PHA, colchicine solution is injected at a ratio of 4 μg: 1g of fish body weight.

[0010] Furthermore, the Carnoy fixative is composed of methanol and glacial acetic acid in a volume ratio of 3:1.

[0011] Beneficial effects of this invention: This method, through gradual heating and two injections of PHA, effectively promotes somatic cell proliferation in the Huangshi catfish, resulting in a large number of chromosome metaphase cells with clear morphology, good dispersion, and complete number. By improving the preparation method, the quality and success rate of chromosome karyotype preparation are significantly enhanced. Attached Figure Description

[0012] Figure 1 These are metaphase chromosomes of the Huangshi catfish, prepared using traditional methods. Figure 2 This is the metaphase of the chromosomes of the Huangshi catfish prepared in Example 1. Detailed Implementation

[0013] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0014] Example 1 An improved method for preparing chromosomes of the Huangshi catfish according to this embodiment is carried out according to the following steps: 1. An improved method for preparing chromosomes of the Huangshi catfish, characterized in that the method for preparing Huangshi catfish chromosomes comprises the following steps: 1. Temporarily hold the Huangshi catfish, allowing the water temperature to rise from 18℃ to 20℃ within 12 hours. Administer the first PHA injection to the Huangshi catfish at a ratio of 6μg:1g of PHA to fish body weight. After 12 hours, when the water temperature rises to 22.5℃, administer the second PHA injection to the Huangshi catfish at a ratio of 8μg:1g of PHA to fish body weight, maintaining the water temperature at 22.5℃. After 3 hours, inject colchicine solution at a ratio of 4μg:1g of colchicine solution to fish body weight. 2. After 3 hours, cut off the gills and tail of the Huangshi catfish and bleed it in water. Dissect the fish, remove the head and kidney, place them in a petri dish containing freshwater fish saline to clean them, remove blood clots, and place them in a culture dish containing physiological saline. Use tweezers to grind the head and kidney tissue, aspirate it into a 10mL centrifuge tube and repeatedly blow it with a pipette, add physiological saline, mix well, centrifuge at 1200r / min for 12min, and discard the supernatant. 3. Add 5 mL of 0.5% KCl hypotonic solution to the product of step 2, mix well, let stand at room temperature for 30 min for hypotonic treatment, add 1 mL of freshly prepared Carnoy fixative, mix well, centrifuge at 1200 r / min for 12 min, discard the supernatant, and mix well. 4. Add 5 ml of Carnoy's fixative to the product from step 3. Use a dropper to mix the product and Carnoy's fixative evenly. Let it stand at room temperature for 20 min, then centrifuge at 1200 r / min for 10 min and discard the supernatant. 5. Repeat step four once, then discard the supernatant; 6. Add 5 ml of Carnoy's fixative to the product from step 5, mix well by pipetting, let stand at room temperature for 10 min, then centrifuge at 1200 r / min for 10 min and discard the supernatant. 7. Add 0.5 ml of Carnoy's fixative to the cell pellet after centrifugation in step 6, mix well to prepare a cell suspension, draw the cell suspension into a dropper, drop it onto a pre-frozen glass slide, and then pass the glass slide over an alcohol lamp flame 2-3 times and let it air dry at room temperature. 8. Stain the dried chromosome slide from step 7 with 10% Giemsa stain for 10 minutes, rinse with tap water, and allow to air dry to complete chromosome specimen preparation, thus obtaining the Huangshi catfish chromosome. Figure 2 As shown.

[0015] Example 2 The difference between this embodiment and Specific Embodiment 1 is that in step one, the Huangshi catfish is injected with a second dose of PHA at a ratio of 6 μg:1g of PHA to fish body weight. Otherwise, it is the same as Specific Embodiment 1.

[0016] Example 3 The difference between this embodiment and specific embodiment 1 is that, in step one, 12 hours after the first injection of PHA, the water temperature rises to 23°C.

[0017] Comparative Example 1 1. Temporarily hold the Huangshi catfish in water at 14℃ and inject them with PHA at a ratio of 10μg:1g of fish body weight; 3 hours later, inject them with colchicine solution at a ratio of 4μg:1g of fish body weight. 2. After 3 hours, cut off the gills and tail of the Huangshi catfish and bleed it in water. Dissect the fish, remove the head and kidney, place them in a petri dish containing freshwater fish saline to clean them, remove blood clots, and place them in a culture dish containing physiological saline. Use tweezers to grind the head and kidney tissue, aspirate it into a 10mL centrifuge tube and repeatedly blow it with a pipette, add physiological saline, mix well, centrifuge at 1000r / min for 10min, and discard the supernatant. 3. Add 5 mL of 0.5% KCl hypotonic solution to the product of step 2, mix well, let stand at room temperature for 30 min for hypotonic treatment, add 1 mL of freshly prepared Carnoy fixative, mix well, centrifuge at 1000 r / min for 10 min, discard the supernatant, and mix well. 4. Add 5 ml of fresh fixative to the product from step 3, mix well by pipetting, let stand at room temperature for 20 min, then centrifuge at 1000 r / min for 10 min and discard the supernatant. 5. Repeat step four once, then discard the supernatant; 6. Add 0.5 ml of Carnoy's fixative to the cell pellet after centrifugation in step 5, mix well to prepare a cell suspension, draw the cell suspension into a dropper, drop it onto a pre-frozen glass slide, and then pass the slide over an alcohol lamp flame 2-3 times and let it air dry at room temperature. VII. Stain the dried chromosome slide from step seven with 10% Giemsa stain for 10 minutes, rinse with tap water, and allow to air dry to complete chromosome specimen preparation, thus obtaining the Huangshi catfish chromosome. Figure 1 As shown.

[0018] like Figures 1-2 It can be seen that the chromosome metaphase phases obtained in Comparative Example 1 were few in number, unevenly dispersed, and unclear in morphology; while the chromosomes prepared in Example 1 were clear in morphology, well dispersed, and complete in number. By improving the preparation method, the quality of chromosome karyotype preparation and the success rate of preparation were effectively improved.

Claims

1. An improved method for preparing chromosomes of the Huangshi catfish, characterized in that... The method for preparing chromosomes from the Huangshi catfish is carried out according to the following steps:

1. During the temporary rearing of Huangshi mantis shrimp, the water temperature is raised from 18℃ to 20℃ within 12 hours, and the mantis shrimp are injected with the first dose of PHA; after 12 hours, the water temperature is raised to 22.5-23℃, and the mantis shrimp are injected with the second dose of PHA, while maintaining the water temperature at 22.5-23℃; after 3 hours, colchicine solution is injected.

2. After 3 hours, cut off the gills and tail of the Huangshi catfish and bleed it in water. Dissect the fish, remove the head and kidney, place them in a petri dish containing freshwater fish saline to clean them, remove blood clots, and place them in a culture dish containing physiological saline. Use tweezers to grind the head and kidney tissue, aspirate it into a 10mL centrifuge tube and repeatedly blow it with a pipette, add physiological saline, mix well, centrifuge at 1200r / min for 12min, and discard the supernatant.

3. Add 5 mL of 0.5% KCl hypotonic solution to the product of step 2, mix well, let stand at room temperature for 30 min for hypotonic treatment, add 1 mL of Carnoy fixative prepared in the ratio of methanol: glacial acetic acid = 3:1, mix well, centrifuge at 1200 r / min for 12 min, discard the supernatant, and mix well.

4. Add 5 ml of Carnoy's fixative to the product from step 3. Use a dropper to mix the product and Carnoy's fixative evenly. Let it stand at room temperature for 20 min, then centrifuge at 1200 r / min for 10 min and discard the supernatant.

5. Repeat step four once, then discard the supernatant; 6. Add 5 ml of Carnoy's fixative to the product from step 5, mix well by pipetting, let stand at room temperature for 10 min, then centrifuge at 1200 r / min for 10 min and discard the supernatant.

7. Add 0.5 ml of Carnoy's fixative to the cell pellet after centrifugation in step 6, mix well to prepare a cell suspension, draw the cell suspension into a dropper, drop it onto a pre-frozen glass slide, and then pass the glass slide over an alcohol lamp flame 2-3 times and let it air dry at room temperature.

8. Stain the dried chromosome slide from step 7 with 10% Giemsa stain for 10 minutes, rinse with tap water, and air dry to complete chromosome specimen preparation, thus obtaining the Huangshi catfish chromosome.

2. The improved method for preparing chromosomes of the Huangshi catfish according to claim 1, characterized in that: In step one, when temporarily holding the Huangshi catfish, the water temperature is raised from 18℃ to 20℃ within 12 hours. The Huangshi catfish is injected with the first dose of PHA at a ratio of 6μg:1g of PHA to fish body weight. After 12 hours, the water temperature is raised to 22.5-23℃, and the Huangshi catfish is injected with the second dose of PHA at a ratio of 6-8μg:1g of PHA to fish body weight. The water temperature is maintained at 22.5-23℃.

3. The improved method for preparing chromosomes of the Huangshi catfish according to claim 1, characterized in that: In step one, 3 hours after the second PHA injection, colchicine solution is injected at a ratio of 4 μg: 1g of fish body weight.

4. The improved method for preparing chromosomes of the Huangshi catfish according to claim 1, characterized in that: Carnos fixative is made of methanol and glacial acetic acid in a volume ratio of 3:1.

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