Identification method of artificial enhancement and release schizothorax wandersoni

Abstract

The invention provides a method for identifying artificially enhanced and released schizothorax brachypoda, which comprises the following steps of: picking micro otolith of the schizothorax brachypoda, polishing, and identifying the artificially enhanced and released schizothorax brachypoda and wild schizothorax brachypoda by comparing the difference between the definition and the width of an otolith growth wheel. The method has the advantages of fast experiment, simple operation and visual identification.

Description

Technical Field

[0001] The invention relates to the technical field of proliferation and release, and in particular to an identification method for artificially propagated and released schizothorax breviscidus. Background Art

[0002] Schizothorax wangchiachii, also known as Burmese fish and sand belly, is a cold-water fish mainly distributed in the Jinsha River and Yalong River systems. It is a unique fish in the upper reaches of the Yangtze River. It has a strong adaptability to high-altitude environment and grows relatively slowly. It was once the main fishing target in the production area and has important economic value.

[0003] Affected by human factors such as hydropower station construction, habitat destruction and overfishing, the resources of short-bearded schizothorax have dropped sharply. In order to protect and restore its wild population, a large number of artificially bred short-bearded schizothorax fry have been released into natural rivers. In the process of proliferation and release, in order to distinguish between artificially released short-bearded schizothorax and wild individuals, chemical markers soaked in fluorescent substances such as alizarin complexes and alizarin red and otolith heat marking are currently used to mark the early released fry on a large scale. However, fluorescent substance soaking and marking can easily lead to the death of a certain proportion of fry and may also cause certain damage to the fish body. Chemical markers and heat marking are also difficult to mark all released fry, so it is impossible to identify all artificially released short-bearded schizothorax through intuitive marking. In view of this, it is necessary to further develop the identification technology of artificially released short-bearded schizothorax to provide a basis for the identification of artificially released populations and the evaluation of release effects. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a method for identifying artificially propagated and released short-whiskered schizothorax, which utilizes the microstructural differences of otoliths to identify artificially propagated and released short-whiskered schizothorax, so as to distinguish artificially propagated and released individuals from wild individuals.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0006] A method for identifying artificially propagated and released short-bearded schizothorax, comprising removing micro-otoliths of the short-bearded schizothorax, grinding them, and distinguishing the artificially propagated and released short-bearded schizothorax from wild short-bearded schizothorax by comparing the differences in clarity and width of the otolith growth rings.

[0007] The growth ring characteristics on the inner and outer sides of the micro-otoliths of artificially released short-bearded schizothorax showed a clear boundary. The outer growth rings were deposited during the growth period in the wild after release, and the inner growth rings were deposited during the breeding period in the fish breeding station before release. The growth rings deposited on the otoliths after release were affected by the wild environment, with reduced clarity, reduced width and increased fluctuations; the growth rings on the micro-otoliths of wild short-bearded schizothorax were more consistent in clarity, uniform in width and with smaller fluctuations.

[0008] The age of the short-bearded schizothorax described in the present invention is 0 to 4 years old. The ring patterns deposited on the otoliths of the short-bearded schizothorax of this age group are usually clearer, which is convenient for observation and detection.

[0009] The identification method of the present invention comprises the following steps:

[0010] A. Use ophthalmic scissors to cut open the skull of the juvenile schizothorax short-bearded and remove the micro-otoliths on the left and right sides;

[0011] B. Use water-abrasive sandpaper to polish the otoliths. After being cleaned with anhydrous ethanol and made transparent with xylene, the polished otoliths are sealed on a glass slide with neutral gum.

[0012] C. Observe the clarity and width characteristics of the micro-otolith growth rings and their changes under an optical microscope, and take photos using a microphotographic system for the measurement of the growth ring width.

[0013] Preferably, in step B, the micro-otolith is fixed on a glass slide using glue, and 600#, 1500#, and 2500# water-abrasive sandpaper are used in sequence to polish along the sagittal section of the otolith, and the otolith is continuously observed under a microscope during the polishing process until it is polished to the growth center of the otolith; then the otolith is turned over and the other side is polished using the same method.

[0014] The present invention takes artificially propagated and released and wild short-bearded schizothorax as research objects, removes the micro-otoliths of the short-bearded schizothorax, polishes them, detects the clarity of the otolith growth rings under an optical microscope, and successfully identifies the artificially propagated and released and wild short-bearded schizothorax individuals by comparing the clarity and width changes of the otolith growth rings of the two.

[0015] The beneficial effects of the present invention are:

[0016] 1. Due to the obvious difference between the artificial breeding environment and the wild natural environment, the environmental differences such as water temperature and food before and after artificial propagation and release cause the clarity and width of the growth rings deposited on the otoliths to change. The present invention finds that the microstructural characteristics such as the clarity and width of the growth rings deposited on the otoliths of artificially bred short-bearded schizothorax individuals before and after release have obvious differences, which can be used to distinguish between artificially propagated and released individuals and wild individuals. The present invention designs and polishes the otoliths of short-bearded schizothorax, uses an optical microscope to detect the microstructural characteristics of the otoliths, and accurately identifies the released individuals and wild individuals of short-bearded schizothorax, which has the advantages of quick experiment, simple operation, and intuitive identification.

[0017] 2. The present invention uses otolith microstructure as an identification feature, which can quickly and accurately distinguish between artificially propagated and released fish and wild short-bearded schizothorax that are difficult to distinguish in morphology. Compared with otolith chemical marking, the identification method of the present invention will not cause harm to the fry artificially raised in the propagation station to be released; compared with otolith thermal marking, the identification method of the present invention does not require the establishment of a specific water temperature control system to raise the fry to be released. The identification method of the present invention can solve the problem that it is difficult to mark all artificially bred short-bearded schizothorax fry during propagation and release, and there is no need to mark the fry before release. It only needs to detect and analyze the otolith microstructure characteristics of the recaptured individuals after release. The operation process is simple and can intuitively identify the difference between released individuals and wild individuals. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Micro-otoliths of Schizothorax breviscar (A, A', B, B') and unlabeled individual micro-otoliths (C, C') marked by Alizarin red immersion. A, B, C: visible light detection, A', B', C': green excitation light detection, arrows: fluorescent labeling.

[0019] Figure 2 Microstructural characteristics of otoliths of artificially propagated and released Schizothorax brachyuratus.

[0020] Figure 3 Otolith microstructural characteristics of artificially cultured short-palpied schizothorax at breeding stations.

[0021] Figure 4 Microstructural characteristics of otoliths of wild schizothorax short-palpied.

[0022] Figure 5 Changes in the width of the micro-otolith growth rings of artificially propagated and released short-palpied schizothorax. The left side of the arrow shows the width of the growth rings deposited during the culture period at the propagation station before release, and the right side shows the width of the growth rings deposited in the wild after release.

[0023] Figure 6 Changes in the width of the micro-otolith growth rings of artificially cultured schizothorax brachyurae at the breeding station.

[0024] Figure 7 Variations in the width of micro-otolith growth rings in wild Schizothorax breviscapus. Specific implementation methods

[0026] In order to explain the technical solution of the present invention more clearly and in detail, the present invention is further described below through relevant embodiments. The following embodiments are only for specific explanation of the implementation method of the present invention, and do not limit the protection scope of the present invention.

[0027] Example 1

[0028] A method for identifying artificially propagated and released short-bearded schizothorax, comprising removing micro-otoliths of the short-bearded schizothorax, grinding them, and distinguishing the artificially propagated and released short-bearded schizothorax from wild short-bearded schizothorax by comparing the differences in clarity and width of the otolith growth rings.

[0029] The growth ring characteristics on the inner and outer sides of the micro-otoliths of artificially released short-bearded schizothorax showed a clear boundary. The outer growth rings were deposited during the growth period in the wild after release, and the inner growth rings were deposited during the breeding period in the fish breeding station before release. The growth rings deposited on the otoliths after release were affected by the wild environment, with reduced clarity, reduced width and increased fluctuations; the growth rings on the micro-otoliths of wild short-bearded schizothorax were more consistent in clarity, uniform in width and with smaller fluctuations.

[0030] The identification method of artificially propagated and released fish of the present invention is suitable for short-bearded schizothorax aged 0 to 4 years old. The ring patterns deposited on the otoliths of short-bearded schizothorax of this age group are usually clearer and easier to observe and detect.

[0031] Example 2

[0032] The identification method of the present invention comprises the following steps:

[0033] A. Use ophthalmic scissors to cut open the skull of the juvenile schizothorax short-bearded and remove the micro-otoliths on the left and right sides;

[0034] B. Use water-abrasive sandpaper to polish the otoliths. After being cleaned with anhydrous ethanol and made transparent with xylene, the polished otoliths are sealed on a glass slide with neutral gum.

[0035] C. Observe the clarity and width characteristics of the micro-otolith growth rings and their changes under an optical microscope, and take photos using a microphotographic system for the measurement of the growth ring width.

[0036] Example 3

[0037] The present invention uses wild, artificially cultivated and artificially released individuals of short-bearded schizothorax as research objects. First, the markers are soaked in alizarin red solution to identify the artificially released individuals, and then the characteristics and changes of the micro-otolith growth rings of the three are observed and compared. The experimental process specifically includes the following steps:

[0038] The first step is to mark the otoliths of the juvenile short-bearded schizothorax to be released by soaking them in an alizarin red solution. Prepare an alizarin red solution (ARS) with a concentration of 20 mg / L. Fast the artificially bred juvenile short-bearded schizothorax for 24 hours, then soak them in the alizarin red solution for 24 hours. The water temperature is maintained at around 16°C. After the soaking, resume feeding the short-bearded schizothorax. Randomly select some of the immersion-marked short-bearded schizothorax individuals, remove the micro-otoliths, and after clearing with anhydrous ethanol and xylene, seal them on a glass slide with neutral gum. Use an upright fluorescence microscope to detect the alizarin red mark on the otolith under visible light and green excitation light. Under green excitation light, the alizarin red mark on the otolith appears red. Under visible light, purple marks can be detected on the otoliths of some individuals. If there is a red fluorescent mark (green excitation light detection) or a purple mark (visible light detection) on the otolith, it means that the alizarin red marking is successful (see Figure 1 ).

[0039] In the second step, the juvenile short-bearded schizothorax with alizarin red successfully marked on the otoliths were released into the wild environment.

[0040] The third step is to collect wild and artificially released short-bearded schizothorax fry with alizarin red marks on their otoliths from the wild, and collect artificially cultured short-bearded schizothorax fry from fish breeding stations.

[0041] In the fourth step, the skulls of wild, cultured and released juvenile short-bearded schizothorax were cut open with ophthalmic scissors, and the micro-otoliths on the left and right sides were removed. The micro-otoliths were washed with anhydrous ethanol, dried and stored in 200 μL centrifuge tubes.

[0042] Step 5: Use crazy glue or other types of glue to fix the micro-otoliths on a glass slide, and use 600#, 1500#, and 2500# water-abrasive sandpaper to polish along the sagittal section of the otoliths. Observe under a microscope during the polishing process until the otolith growth center is polished. Then turn the otoliths over and polish the other side in the same way. The polished otoliths are cleaned with anhydrous ethanol and made transparent with xylene, and sealed with neutral gum on a glass slide for testing.

[0043] Step 6: The characteristics and changes of the otolith growth rings of wild, cultured and released juvenile schizothorax were examined under an Olympus BH2 optical microscope, and the otolith microstructures were photographed using a microphotography system (Leica DM 4B) (see Figure 2-4 ), analyzed and compared the clarity and width of the otolith growth rings of artificially propagated, artificially cultured and wild schizothorax, and counted the number of growth rings corresponding to different growth ring clarity and width (see Figure 5-7 ).

[0044] Depend on Figure 3 and Figure 4It can be seen that the clarity of the growth rings on the micro-otoliths of artificially cultured and wild Schizothorax brevis is relatively consistent, and the growth rings on the micro-otoliths of artificially cultured individuals are wider than those of wild individuals. Figure 2 It can be seen that the characteristics of the micro-otolith growth rings of artificially released short-bearded schizothorax have obvious changes in clarity and width, and there is a clear boundary between the inner and outer sides. The growth rings on the outer side of the micro-otoliths are deposited during the growth period in the wild after release, and the growth rings on the inner side are deposited during the breeding period in the breeding station before release. This difference in the characteristics of the otolith growth rings before and after release is caused by the large difference between the breeding conditions in the breeding station before release and the wild environment after release. Therefore, individuals with obvious clarity and width boundaries on the growth rings on the otoliths can be judged as artificially released individuals, and those with a small change in the width of the growth rings and basically the same clarity can be judged as wild individuals.

[0045] A microscope was used to take photos of the otolith microstructure, and then an otolith image analysis system was used (the analysis method refers to invention patent CN1361475A) to measure the width of the otolith growth rings. The clarity and width changes of the otolith growth rings of artificially propagated and released, artificially cultured and wild short-bearded schizothorax were analyzed and compared, and the number of growth rings corresponding to different growth ring clarity and width were counted.

[0046] In the present invention, the number of clear growth rings on the outer side of the alizarin red fluorescent mark on the otolith of the released individual is 85±4, the average width is 2.278±0.538μm, and the fluctuation range is 1.155-4.919μm. The 87th to 92nd growth rings on the outer side of the fluorescent mark are unclear, and the number of unclear growth rings is about 5. The clarity and width of the growth rings after the 92nd on the outer side of the fluorescent mark are significantly reduced, and the average width is 1.674±0.546μm, and the fluctuation range is 0.633-5.029μm. The clarity of the growth rings of the otoliths of artificially cultivated individuals is relatively high, the number of growth rings is 145±5, the width is 2.542±0.571μm, and the fluctuation range is 0.98-4.889μm. The clarity of the otolith growth rings of wild individuals is relatively high, with 75±3 growth rings, a width of 2.337±0.316μm, and a fluctuation range of 1.704-4.191μm. The experiment showed that the clarity of the growth rings deposited on the micro-otoliths of the short-bearded schizothorax was significantly reduced and the width became smaller within about 5 days after release, which was significantly different from the characteristics of the otolith growth rings of artificially raised and wild individuals. Therefore, based on the differences in the characteristics of the otolith growth rings, the released individuals can be identified.

[0047] The above examples only express the specific implementation of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for identifying artificially propagated and released Schizothorax short-palpied, characterized in that: The micro-otoliths of the short-bearded schizothorax were removed and polished, and the differences in clarity and width of the otolith growth rings were compared to identify the artificially propagated and released short-bearded schizothorax from the wild.

2. The identification method of artificially propagated and released Schizothorax brevis according to claim 1, characterized in that: The growth ring characteristics on the inner and outer sides of the micro-otoliths of artificially released short-bearded schizothorax showed a clear boundary. The outer growth rings were deposited during the growth period in the wild after release, and the inner growth rings were deposited during the breeding period in the fish breeding station before release. The growth rings deposited on the otoliths after release were affected by the wild environment, with reduced clarity, reduced width and increased fluctuations; the growth rings on the micro-otoliths of wild short-bearded schizothorax were more consistent in clarity, uniform in width and with smaller fluctuations.

3. The identification method of artificially propagated and released Schizothorax brevis according to claim 1, characterized in that: The age of the short-bearded schizothorax is 0 to 4 years old.

4. The identification method of artificially propagated and released Schizothorax brevis according to claim 1, characterized in that: The following steps are involved: A. Use ophthalmic scissors to cut open the skull of the juvenile schizothorax short-bearded and remove the micro-otoliths on the left and right sides; B. Use water-abrasive sandpaper to polish the otoliths. After being cleaned with anhydrous ethanol and made transparent with xylene, the polished otoliths are sealed on a glass slide with neutral gum. C. Observe the clarity and width characteristics of the micro-otolith growth rings and their changes under an optical microscope, and take photos using a microphotographic system for the measurement of the growth ring width.

5. The identification method of artificially propagated and released Schizothorax brevis according to claim 1, characterized in that: In the step B, the micro-otolith is fixed on a glass slide using glue, and 600#, 1500#, and 2500# water-abrasive sandpaper are used to polish along the sagittal section of the otolith in sequence, and the otolith is continuously observed under a microscope during the polishing process until the otolith growth center is polished; then the otolith is turned over and the other side is polished using the same method.

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