CN112729982A - Preparation method of material for identifying age of shark - Google Patents
Preparation method of material for identifying age of shark Download PDFInfo
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- CN112729982A CN112729982A CN202011520911.9A CN202011520911A CN112729982A CN 112729982 A CN112729982 A CN 112729982A CN 202011520911 A CN202011520911 A CN 202011520911A CN 112729982 A CN112729982 A CN 112729982A
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- 239000000463 material Substances 0.000 title claims abstract description 33
- 241000251730 Chondrichthyes Species 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000000227 grinding Methods 0.000 claims abstract description 26
- 210000002808 connective tissue Anatomy 0.000 claims abstract description 21
- 239000012153 distilled water Substances 0.000 claims abstract description 20
- 238000002791 soaking Methods 0.000 claims abstract description 20
- 238000005406 washing Methods 0.000 claims abstract description 17
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 12
- 238000009835 boiling Methods 0.000 claims abstract description 7
- 238000004140 cleaning Methods 0.000 claims abstract description 7
- 238000000605 extraction Methods 0.000 claims abstract description 7
- 238000007710 freezing Methods 0.000 claims abstract description 7
- 230000008014 freezing Effects 0.000 claims abstract description 7
- 210000003205 muscle Anatomy 0.000 claims abstract description 7
- 238000000643 oven drying Methods 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 238000004043 dyeing Methods 0.000 claims description 6
- 238000005498 polishing Methods 0.000 claims description 5
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000000853 adhesive Substances 0.000 claims description 3
- 230000001070 adhesive effect Effects 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 239000010413 mother solution Substances 0.000 claims description 2
- 210000003462 vein Anatomy 0.000 abstract description 11
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 238000007789 sealing Methods 0.000 abstract 1
- 241000251468 Actinopterygii Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a preparation method of a material for identifying the age of shark, which comprises the following steps: (1) extraction: taking about 3-5 vertebra from the first dorsal fin, and freezing and storing; (2) cleaning: removing muscle and connective tissue from vertebra, washing with distilled water, decocting vertebra in boiling water for 5-10min to remove hard-to-remove connective tissue, and placing in 30% H solution if connective tissue is still attached2O2Soaking in the solution for about 1-2 h, soaking in distilled water, washing repeatedly, and oven drying; (3) coarse grinding: the two sides were ground to approximately the center using 120grit waterproof and wear resistant sandpaper on a metallographic grinder to form vertebral bone slices approximately 3-5mm thick. The invention polishes the longitudinal section of the long-fin shark spinal vertebra, applies force uniformly, polishes the polished spinal bone slice and seals the slice by the sealing agent, and ensures that the wheel veins can be kept for a longer time.
Description
Technical Field
The invention relates to a preparation method of a material for identifying the age of marine organisms, in particular to a preparation method of a material for identifying the age of shark.
Background
The long-fin shark (Carcharinus longibranus longimanus) belongs to the family Carcharinuriformes (Carcharinuriformes) of the family Carcharinuriformes (Carcharinuridae), is a large-scale medium-upper-layer shark, is positioned at the top of a food chain, is one of the most abundant species in tropical sea area resources, and is also one of the common tuna longline fishing and fishing species. However, with the increasing of over-fishing and the worsening of marine environment in recent years, the resource amount of long-fin shark has seriously declined and is now classified as endangered species by the international union of natural protection (IUCN).
The fish age information is the basis for fishery resource assessment, and accurate age and growth information is helpful for predicting fish resource variation in population dynamics research. Vertebra bone has the characteristic of stably storing growth information, and is the most effective material for researching the age and growth of shark; the vertebra is of a biconcave type, and the upper wheel veins of the vertebra are characterized in that wide opaque wheel veins and narrow semitransparent wheel veins are alternately arranged and are in concentric circles. The opaque ring veins are formed in warmer spring and summer with faster growth, the semi-transparent ring veins are formed in autumn and winter with lower temperature and slow growth, and the junction of the opaque ring veins in the current year and the semi-transparent ring veins in the next year is the annual ring.
The long-fin shark has the characteristics of slow growth, late sexual maturity, long breeding period, long service life and the like, so that the capability of bearing fishing pressure is extremely low, and the research on the age and growth of the long-fin shark is beneficial to evaluating the change of the resource condition of the long-fin shark. The vertebra is used as a growth information carrier, which can stably store the growth information input by the body and reveal the growth and development process. However, as fish ages, the radius of the vertebra increases, and the ring tracks near the outer edge become more dense and even overlap, so that the choice of the age identification method is important.
Disclosure of Invention
The invention provides a preparation method of a material for identifying the age of a shark, aiming at enabling the ring lines on the complete vertebra to be clearer through a dyeing method and solving the problems that a small vertebra slice is not easy to grind, is easy to damage in the grinding process, is not high in ring line definition after grinding and the like.
The invention is realized by the following technical scheme:
a preparation method of a material for identifying the age of shark is characterized by selecting a vertebra material, and then comprising the following steps:
(1) extraction: taking about 3-5 vertebra from the first dorsal fin, and freezing and storing;
(2) cleaning: removing muscle and connective tissue from vertebra, washing with distilled water, decocting vertebra in boiling water for 5-10min to remove hard-to-remove connective tissue, and placing in 30% H solution if connective tissue is still attached2O2Soaking in the solution for about 1-2 h, soaking in distilled water, washing repeatedly, and oven drying;
(3) coarse grinding: roughly grinding the two sides of the vertebra by using 120-grit waterproof wear-resistant abrasive paper on a metallographic grinder until the two sides are respectively ground to be close to the central position to form a vertebra slice with the thickness of about 3-5 mm;
preferably, different sizes of vertebra use different material, and the step (1) extracts larger vertebra material.
Preferably, after the coarse grinding in step (3), the following two steps are added:
fixing: adhering the longitudinal section to the glass slide with strong fast adhesive, and the central surface of the vertebral body facing downwards;
grinding: and (3) polishing the slices by using 600grits and 1200grits of waterproof wear-resistant abrasive paper, observing the grinding degree under a transmitted light dissecting mirror at any time until the wheel pattern is clear and the grinding thickness of the slices is about 1mm, and placing the samples into a sample box for age identification.
Preferably, the vertebra material is selected from the vertebra with unclear grinding wheel veins and the material with the vertebra which is small and difficult to be directly ground,
preferably, the preparation method of the material which is hard to directly grind the vertebra with the unclear grinding wheel veins and the vertebra with small grinding wheel veins comprises the following steps:
(1) extraction: taking about 3-5 vertebra from the first dorsal fin, and freezing and storing;
(2) cleaning: removing muscle and connective tissue from vertebra, washing with distilled water, decocting vertebra in boiling water for 5-10min to remove hard-to-remove connective tissue, and placing in 30% H solution if connective tissue is still attached2O2Soaking in the solution for about 1-2 h, soaking in distilled water, washing repeatedly, and oven drying;
(3) dyeing: and (2) soaking the dried whole vertebra in distilled water for 12h, rinsing with alcohol for 2 times, then washing with distilled water, rinsing with 4% KOH for 3 times every 16h, finally placing in alizarin red solution with the mass concentration of 0.01% and 1% KOH as mother solution for 12h, taking out, soaking for 1h by using 1% KOH solution, and finally drying in the air.
Preferably, the diameter of the larger vertebral bone material is greater than or equal to 10mm,
preferably, the vertebra is smaller than an individual with a diameter <10mm of material that is difficult to grind directly.
The invention has the beneficial effects that:
the method comprises the steps of preparing materials for identifying the age of a long fin real shark of a larger vertebra sample (the diameter is more than or equal to 10mm), a smaller vertebra sample (the diameter is less than 10mm) and a sample with unclear wheel lines, finding that most of long fin real shark samples of the smaller vertebra with the diameter less than 10mm and the vertebral bone samples with unclear wheel lines after grinding are suitable for preparing the materials by using a complete vertebral bone dyeing method according to experiments, and determining that alizarin red is optimally dyed by the experiments; and the vertebra with the diameter more than or equal to 10mm is larger, the ring lines can be overlapped at the edge, slice observation is carried out selectively, and the material effect of longitudinal slice preparation is better because the vertebra material has partial cartilage.
The flaking of the long-fin shark spinal bone is a complicated and fine process, and the quality of flaking effect is directly related to the accuracy and reliability of age identification; stain treatment can deepen the clarity of the annual rings, and is particularly important for the accuracy of identification. The invention makes the ring lines on the complete vertebra clearer by a dyeing method, solves the problems that smaller vertebra slices are not easy to grind, are easy to damage in the grinding process, and have low ring line definition after grinding, and the like, provides a preparation method which can be quickly and accurately applied to the material for identifying the vertebra ages of long-fin shark vertebrae with different sizes, greatly shortens the identification time of the prepared material, ensures higher identification success rate, and simultaneously enables the ring lines of the grinded vertebrae to be kept for a longer time.
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, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic view of the shape of a long-fin shark spinal bone of the invention with a diameter of 10mm or more;
FIG. 2 is a schematic diagram of a longitudinal section of a long fin shark spinal bone morphology with a diameter of 10mm or more according to the present invention;
FIG. 3 is a graph showing the staining of spinal bones by a long fin shark of less than 10mm in diameter according to the present invention;
the symbols in the figures represent:
1. a vertebral body; 2. the center of the vertebral body; 3. a central plane of the vertebral body; 4. and (5) growing the wheel pattern.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below by combining the specific drawings.
The invention discloses a preparation method of a material for identifying the age of shark, wherein different materials are used for vertebrae with different sizes;
referring to fig. 1 and 2, the preparation method of the material of the large vertebra (the diameter is more than or equal to 10mm) comprises the following steps:
(1) extraction: taking about 3-5 vertebra from the first dorsal fin, and freezing and storing;
(2) cleaning: removing muscle and connective tissue from vertebra, washing with distilled water, decocting vertebra in boiling water for 5-10min to remove hard-to-remove connective tissue, and placing in 30% H solution if connective tissue is still attached2O2Soaking in the solution for about 1-2 h, soaking in distilled water, washing, and oven drying to make vertebra clearly visible in vertebral body 1 and vertebral body center 2;
(3) coarse grinding: roughly grinding the two surfaces of the vertebra by using 120-grit waterproof wear-resistant abrasive paper on a metallographic grinder until the two surfaces are close to the central position respectively to form a vertebra slice with the thickness of about 3-5mm to form a centrum central surface 3;
fixing: adhering the longitudinal section to the glass slide with strong fast adhesive, and the central surface of the vertebral body facing downwards;
grinding: and (3) polishing the slices by using 600grits and 1200grits of waterproof wear-resistant abrasive paper, observing the polishing degree of the slices under a transmitted light dissecting mirror at any time until the wheel grains are clear, growing the wheel grains 4, and optimally polishing the slices to the thickness of about 1mm, and putting the samples into a sample box for age identification.
Referring to FIG. 3, the method for preparing small vertebrae (diameter <10mm) and grinding of the spinal material with unclear striations comprises the following steps:
(1) extraction: taking about 3-5 vertebra from the first dorsal fin, and freezing and storing;
(2) cleaning: removing muscle and connective tissue from vertebra, washing with distilled water, decocting vertebra in boiling water for 5-10min to remove hard-to-remove connective tissue, and placing in 30% H solution if connective tissue is still attached2O2Soaking in the solution for about 1-2 h, soaking in distilled water, washing repeatedly, and oven drying;
(3) dyeing: and (2) soaking the dried whole vertebra in distilled water for 12h, rinsing with alcohol for 2 times, then washing with distilled water, rinsing with 4% KOH for 3 times every 16h, finally placing in alizarin red solution with the mass concentration of 0.01% and 1% KOH as mother liquor for 12h, taking out, soaking for 1h with 1% KOH solution, and finally airing, wherein the grown ring 4 is clearly visible.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A preparation method of a material for identifying the age of shark is characterized in that the method comprises the following steps:
(1) extraction: taking about 3-5 vertebra from the first dorsal fin, and freezing and storing;
(2) cleaning: removing muscle and connective tissue from vertebra, washing with distilled water, decocting vertebra in boiling water for 5-10min to remove hard-to-remove connective tissue, and placing in 30% H solution if connective tissue is still attached2O2Soaking in the solution for about 1-2 h, soaking in distilled water, washing repeatedly, and oven drying;
(3) coarse grinding: the two sides were ground to approximately the center using 120grit waterproof and wear resistant sandpaper on a metallographic grinder to form vertebral bone slices approximately 3-5mm thick.
2. The method of claim 1, wherein different sizes of vertebrae are used and the step (1) extracts larger vertebrae material.
3. The method for preparing a long-fin shark age identification material as claimed in claim 1, wherein the following two steps are added after the coarse grinding in step (3):
(31) fixing: adhering the longitudinal section to the glass slide with strong fast adhesive, and the central surface of the vertebral body facing downwards;
(32) grinding: and (3) polishing the slices by using 600grits and 1200grits of waterproof wear-resistant abrasive paper, observing the grinding degree under a transmitted light dissecting mirror at any time until the wheel pattern is clear and the grinding thickness of the slices is about 1mm, and placing the samples into a sample box for age identification.
4. The method of claim 1, wherein the vertebra material is selected from vertebra with unclear grinding ring and vertebra with small difficulty in direct grinding.
5. The method of claim 1, wherein the method for preparing a material for identifying the age of shark, comprises the following steps:
(1) extraction: taking about 3-5 vertebra from the first dorsal fin, and freezing and storing;
(2) cleaning: removing muscle and connective tissue from vertebra, washing with distilled water, decocting vertebra in boiling water for 5-10min to remove hard-to-remove connective tissue, and placing in 30% H solution if connective tissue is still attached2O2Soaking in the solution for about 1-2 h, soaking in distilled water, washing repeatedly, and oven drying;
(3) dyeing: and (2) soaking the dried whole vertebra in distilled water for 12h, rinsing with alcohol for 2 times, then washing with distilled water, rinsing with 4% KOH for 3 times every 16h, finally placing in alizarin red solution with the mass concentration of 0.01% and 1% KOH as mother solution for 12h, taking out, soaking for 1h by using 1% KOH solution, and finally drying in the air.
6. The method of claim 2, wherein the diameter of the larger vertebral bone material is greater than or equal to 10 mm.
7. The method of claim 4, wherein the vertebra is smaller and the diameter of the material difficult to grind directly is less than 10 mm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011520911.9A CN112729982A (en) | 2020-12-21 | 2020-12-21 | Preparation method of material for identifying age of shark |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011520911.9A CN112729982A (en) | 2020-12-21 | 2020-12-21 | Preparation method of material for identifying age of shark |
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| CN112729982A true CN112729982A (en) | 2021-04-30 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104931665A (en) * | 2015-05-26 | 2015-09-23 | 上海海洋大学 | Construction method of vertebral growth characteristic analytical model for mackerel pike |
| CN105806678A (en) * | 2016-05-12 | 2016-07-27 | 上海海洋大学 | Preparation method of material for identifying age of blue shark |
| CN106645150A (en) * | 2016-11-25 | 2017-05-10 | 上海海洋大学 | Method for determining ages of shrimps and crabs by using eyestalk |
| CN207366823U (en) * | 2017-10-23 | 2018-05-15 | 中国水产科学研究院长江水产研究所 | A kind of infuser device for identifying the fish vertebrae age |
| CN110495445A (en) * | 2019-09-20 | 2019-11-26 | 河南师范大学 | A kind of method of the Yellow River carp postlarva intermuscular bone dyeing |
-
2020
- 2020-12-21 CN CN202011520911.9A patent/CN112729982A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104931665A (en) * | 2015-05-26 | 2015-09-23 | 上海海洋大学 | Construction method of vertebral growth characteristic analytical model for mackerel pike |
| CN105806678A (en) * | 2016-05-12 | 2016-07-27 | 上海海洋大学 | Preparation method of material for identifying age of blue shark |
| CN106645150A (en) * | 2016-11-25 | 2017-05-10 | 上海海洋大学 | Method for determining ages of shrimps and crabs by using eyestalk |
| CN207366823U (en) * | 2017-10-23 | 2018-05-15 | 中国水产科学研究院长江水产研究所 | A kind of infuser device for identifying the fish vertebrae age |
| CN110495445A (en) * | 2019-09-20 | 2019-11-26 | 河南师范大学 | A kind of method of the Yellow River carp postlarva intermuscular bone dyeing |
Non-Patent Citations (1)
| Title |
|---|
| 王浩展: "热带大西洋拟锥齿鲨的生物学特征及其资源分布与环境关系的研究", 中国优秀硕士学位论文全文数据库 农业科技辑, 15 March 2018 (2018-03-15), pages 17 - 20 * |
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