A method for individual marking of giant freshwater prawns

Abstract

A method for individual tagging of giant freshwater prawns includes the following steps: S1, Pre-tag preparation: Purchasing implantable glass tube-encapsulated RFID chip tags, matching syringes or injection equipment, animal tissue adhesive, and thin plastic film discs; Temporarily raising the prawns to be tagged to a length of more than 3cm, from the base of the eyestalk to the end of the telson; S2, Preparing the prawns to be tagged: Removing the prawns to be tagged from the temporary holding tank and placing them in water at 16-22℃ to await tagging; In summary, the tagging method of the present invention greatly increases the overall tag retention rate and survival tag retention rate without changing the mortality rate, making it possible to tag giant freshwater prawns with implantable tags, and allowing the tags to accompany the prawns for a long period of time, making it possible to apply implantable tags to long-term production experiments such as breeding.

Description

Technical Field

[0001] This invention belongs to the field of crustacean individual marking technology, specifically relating to a method for marking individual giant freshwater prawns. Background Art

[0002] The giant freshwater prawn (Macrobrachium rosenbergii) is one of the world's three major farmed freshwater shrimp species. This shrimp is characterized by rapid growth, a wide diet, low disease incidence, high-quality meat, and a short farming cycle, giving it excellent economic value. As an important component of my country's freshwater aquaculture industry, the giant freshwater prawn has a complete industrial chain and significant economies of scale, making a substantial contribution to increasing fishery efficiency and fishermen's income. However, with the explosive growth of the industry, some major industrial problems have become increasingly prominent. Among them, the problem of genetic degradation caused by unscientific methods of seed preservation, introduction, seedling production, and farming is particularly prominent. The cultivation of "superior breeds" is key to solving this major industrial problem.

[0003] In recent years, both domestic and international breeding theories, technologies, and methodologies have been continuously updated and iterated. The latest generation of breeding systems can be roughly summarized as molecular-assisted design breeding, which utilizes animal genetic information to assist breeders in making targeted breeding plans to accelerate the overall breeding process. This system requires that the phenotypic traits, genotype information, and other production information of the individuals to be bred must be mapped one-to-one to the individual, ensuring data traceability and the retrieval of tagged shrimp. Therefore, the individual tagging and identification methods for the individuals to be bred are a prerequisite for the operation of the entire system.

[0004] In other species, methods for marking and identifying individuals are not difficult to implement. Stem tags and cultivation vessel labels in crops perfectly solve the problem of individual marking and identification; ear tags, leg tags, and neck rings in livestock and poultry can also conveniently mark and identify individuals. However, for rhythmically molting crustaceans, methods for marking and identifying individuals are a significant challenge.

[0005] Currently, there are two main theoretically feasible methods for marking crustaceans: (1) injecting pigment into the body and using different injection sites and colors to distinguish individuals; (2) raising each individual in an independent grid and marking the grid to distinguish individuals within the grid. The problem with method (1) is that the limited color combinations restrict the maximum number of distinguishable marks. Especially when too many color types are used, it is difficult to read similar colors under the animal's shell. Furthermore, as individuals grow, color marks are torn and moved, further increasing the difficulty of identification. Currently, mature commercial products based on this method support a maximum number of distinguishable marks of no more than 1,000 and are only used for distinguishing multiple groups. The problem with method (2) is that limited production facilities restrict the maximum number of distinguishable marks. The cramped grids will affect the normal growth of the individuals to be bred (mortality rate is much higher than that of temporary holding in ponds), and the subsequent production management of the grids is also extremely cumbersome (such as feeding each grid and vacuuming up waste). This method is only applied to distinguish individuals in short-term production stages such as overwintering. A long-lifecycle, high recognition rate, low de-labeling rate, and strong practicality for production individual marking method of giant freshwater prawns is a key technology urgently needed in giant freshwater prawn breeding and other breeding scenarios. Summary of the Invention

[0006] This invention aims to provide a method for marking individual giant freshwater prawns, solving the technical problem in the prior art that limits the maximum number of distinguishable marks due to limited color combinations or limited production facilities.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a method for individual marking of giant freshwater prawns, comprising the following steps:

[0008] S1. Pre-marking preparation: Purchase implantable glass tube-encapsulated RFID chip tags, matching syringes or injection equipment, animal tissue adhesive, and thin plastic film discs; temporarily raise the shrimp to be marked to a length of more than 3cm, which is from the base of the eyestalk to the end of the telson.

[0009] S2. Preparation of shrimp to be tagged: Remove the shrimp to be tagged from the holding tank and place them in water at 16-22℃ to wait for tagged;

[0010] S3, Tag Loading: The operator wears gloves and holds a label syringe to load the implantable glass tube-encapsulated RFID chip tag into the needle;

[0011] S4. Adhesive for injection needle: After attaching the nozzle of the animal tissue adhesive, squeeze out a small amount of adhesive onto the syringe needle that has been labeled, so that the adhesive completely covers the needle hole for 2-3 mm.

[0012] S5. Tag injection: Remove the shrimp to be labeled from the water, gently and evenly dry the shrimp body with an absorbent cloth, hold the cephalothorax and tail with one hand, and inject a glass tube label into the front half of the shrimp tail.

[0013] S6. Needle hole injection: After the syringe needle is pulled out, seal the needle hole on the shrimp shell with animal tissue glue, making sure the glue covers the needle hole.

[0014] S7. Pinhole sealing: Stick the prepared thin plastic film disc to the sealed pinhole, and gently rub the thin plastic film disc until it is completely and firmly stuck.

[0015] S8. Settling gel: Settle the tagged shrimp in the gel, then return the tagged shrimp to the temporary container.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides a method for individual tagging of giant freshwater prawns that can accompany the prawns for a long lifespan, has a high recognition rate, a low tag removal rate, and strong practicality in production. It can be quickly integrated into the molecular-assisted design breeding system of giant freshwater prawns, achieving the correspondence between various breeding data and individuals in the breeding process. This invention can also play a key role in other production scenarios of giant freshwater prawns. The tagging method described in this invention greatly increases the overall tag retention rate and the survival tag retention rate without changing the mortality rate, making it possible to tag giant freshwater prawns with implanted tags, and allowing the tags to accompany the prawns for a long period of time, making it possible to apply implanted tags to long-term production experiments such as breeding. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0018] Figure 1 Line graph showing changes in label retention rate and label rejection rate after labeling. Detailed Implementation

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Specific embodiment 1 of the individual marking method for giant freshwater prawns provided by the present invention:

[0022] like Figure 1 As shown, a method for individual marking of giant freshwater prawns includes the following steps:

[0023] S1. Pre-marking preparation: Purchase implantable glass tube-encapsulated RFID chip tags, matching syringes or injection equipment, animal tissue adhesive, and thin plastic film discs. The diameter of the implantable glass tube-encapsulated RFID chip tags is 1.25mm and the length is 7mm. The animal tissue adhesive is cyanoacrylate adhesive or other alternatives, but it must meet the "animal use" safety level. The thin plastic film discs are made of PE plastic bags (0.01mm to 0.05mm thickness is acceptable) cut into discs with a diameter of 5mm to 7mm. Temporarily raise the shrimp to be marked to a length of more than 3cm, which is from the base of the eyestalk to the end of the telson. Preferably, the water temperature and feed level can be appropriately reduced before marking to slow down the shrimp's metabolic level and adjust the shrimp to the intermolting period.

[0024] S2. Preparation of shrimp to be tagged: Remove the shrimp to be tagged from the holding tank and place them in water at 16-22℃ to wait for tagged;

[0025] S3, Tag loading: The operator wears gloves and holds a labeled syringe, pushes and pulls the empty needle several times to ensure smooth pushing and pulling, and loads the implantable glass tube-encapsulated RFID chip tag into the needle.

[0026] S4. Adhesive for injection needle: After attaching the nozzle of the animal tissue adhesive, squeeze out a small amount of adhesive onto the syringe needle that has been labeled, so that the adhesive completely covers the needle hole for 2-3 mm.

[0027] In subsequent steps, please note that animal tissue glue will rapidly denature and solidify upon contact with animal tissue fluid. Water will dilute the animal tissue glue, making it even more difficult to solidify. Therefore, avoid contact with water during glue handling. Furthermore, only when the glue at the front and back of the label comes into contact with the tissue fluid inside the shrimp will it help secure the label. During other operations, avoid contact with the tissue fluid to prevent premature solidification. Regularly wipe gloves, syringes, and work surfaces to ensure they are free of water, tissue fluid, and contaminants. Replace gloves and syringes if necessary.

[0028] S5. Tag Injection: Remove the shrimp to be labeled from the water, gently and evenly dry the shrimp with an absorbent cloth, hold the cephalothorax and tail with one hand, and inject the glass tube label into the anterior half of the shrimp's tail (from the first to the third abdominal segment). Experiments have verified that the following two needle insertion and injection methods are practical for production:

[0029] A. Insert the needle into the middle of the side of the first to third abdominal segments, with the needle direction at 90° or slightly less than 90° to the central axis of the shrimp body (for shrimp to be labeled with a cross-sectional diameter of less than 10mm, inserting the needle at a full 90° angle will easily cause the needle tip to come out from the opposite side, so for this type of shrimp, the needle should be inserted at a less than 90° angle). Stop the needle when the tip is about to the central axis, and gently and steadily push it into the glass tube label.

[0030] B: Gently bend the shrimp's tail with one hand until it is fully arched (as shown in the picture), insert the needle at the junction of the third and fourth abdominal segments, with the needle direction slightly parallel to the central axis of the shrimp's cephalothorax and towards the cephalothorax. Stop inserting the needle after it enters the first abdominal segment (do not go beyond the first abdominal segment to prevent it from entering the cephalothorax and damaging internal tissues and organs), and gently and steadily push the glass tube label in.

[0031] S6. Needle hole injection: After the syringe needle is pulled out, seal the needle hole on the shrimp shell with animal tissue glue, making sure the glue covers the needle hole.

[0032] S7. Pinhole sealing: Stick the prepared thin plastic film disc to the sealed pinhole, and gently rub the thin plastic film disc until it is completely and firmly stuck.

[0033] S8. Allow gel to set: After gelation, return the tagged shrimp to the temporary container.

[0034] It should be noted that animal tissue glue will rapidly denature and solidify upon contact with animal tissue fluid. Under normal conditions, after sealing the pinhole with plastic film and animal tissue glue for 15 seconds, the tagged shrimp can be placed back into the temporary container. If the glue becomes wet and the gelation time must be extended, the shrimp's cephalothorax can be wrapped with damp gauze to extend the settling time to 2 to 3 minutes. Generally, the plastic film covering the pinhole will not fall off on its own after the shrimp is placed back into the temporary container. If the plastic film falls off immediately after being placed in the temporary container, check whether the gelation was properly settling. Select several tagged shrimp and squeeze the shrimp with the label wrapped around it but away from the pinhole to check if the label is easily squeezed out. If so, the gelation steps need to be adjusted until the label is properly fixed inside the shrimp.

[0035] Regarding the post-treatment management of tagged shrimp: In the later stages of production management, tagged shrimp should still be kept at a low temperature (below 26℃ but not too low) for several days to help wounds heal. Afterwards, the temperature can be raised normally, and feeding can begin, awaiting the first molt after tagged shrimp. If, during the first molt after tagged shrimp, a large number of tags are observed being squeezed out of the pinholes, it is necessary to review the previous steps, including the tagging location, the use of animal tissue glue, and the sealing of the pinholes with plastic film, to identify any problems and correct them to reduce the final untagged rate.

[0036] Tagged shrimp identification: Microchips and corresponding receivers conforming to the ISO 11784 code structure and ISO 11785 technical standards using contactless radio frequency identification (RFID) technology are acceptable (such as the FDX-B format under the ISO11784 / 85 standard commonly used in my country).

[0037] During the research and development phase of this invention, the effects of different injection sites, pre- and post-injection marking, and wound closure procedures on the label removal rate were investigated. To obtain the trend of the label removal rate over time, the following data were recorded for different time periods:

[0038] (1) Number of individuals marked, which is the number of living individuals still in the body at that time point;

[0039] (2) Number of individuals that have lost their markers, which is the number of living individuals whose markers are no longer in the body at that time point;

[0040] (3) Number of dead individuals, the number of dead shrimp at this point in time (without recording whether the dead shrimp had already been removed from the label);

[0041] (4) Retention rate (overall), number of retained individuals ÷ total number of individuals;

[0042] (5) Disqualification rate (overall), number of disqualified individuals ÷ total number of individuals;

[0043] (6) Mortality rate, number of dead individuals ÷ total number of individuals;

[0044] (7) Number of surviving individuals, the number of living individuals at this time point (= number of individuals with tags + number of individuals without tags);

[0045] (8) Retention rate (survival), number of retained individuals ÷ number of surviving individuals;

[0046] (9) Disqualification rate (survival), number of disqualified individuals ÷ number of surviving individuals.

[0047] Table 1: Number of tags retained, tags removed, and deaths at different stages after marking.

[0048]

[0049]

[0050] (Note: Tail mark, injection mark on the tail; chest mark, injection mark on the third abdominal segment; whether there are auxiliary marks, and whether glue and plastic film are used to fix and seal the marks and wounds.)

[0051] Table 2 shows the retention, removal, and mortality rates at different stages after labeling.

[0052]

[0053] As shown in Table 2, 90 days after the shrimp tails were tagged, the overall tagged retention rate was less than 25%, and the survival rate was approximately 40%. To guarantee the final number of tagged individuals and the amount of data, the actual number of shrimp farmed would need to be more than four times the designed number, which would be prohibitively expensive. Furthermore, more than half of the surviving shrimp had already detached from the tags, affecting the speed of data collection. Additionally, using molecular methods to confirm the phylogenetic relationships of all detached shrimp would be prohibitively costly, ultimately necessitating the discarding of all detached shrimp. In conclusion, tagged shrimp tails are not practical for production.

[0054] No auxiliary measures are applied during tag implantation, such as Figure 1As shown, the percentage of detached individuals increases from 4% to 15% after 5 days to 44% to 59% after 90 days. The number of detached individuals continues to rise rapidly over 90 days. This phenomenon can be explained by the fact that each molt of the shrimp can lead to detachment. Without intervention, molting significantly increases the likelihood of detachment. As the rearing time increases, the overall detachment rate of the tagged population increases rapidly. When the survival detachment rate reaches 30% or higher, the shrimp is practically no longer suitable for production.

[0055] The tagging method described in this invention achieves an overall tag retention rate of approximately 50% after 90 days of tag implantation (with a mortality rate of approximately 40% and an overall tag detachment rate of approximately 10%, meaning most of the remaining shrimp are dead), and a tag retention rate of approximately 83% among the surviving shrimp. Compared to tagging methods at other sites and those without auxiliary measures, the tagging method described in this invention significantly increases the overall tag retention rate and the survival tag retention rate with almost no change in mortality rate. This makes it possible to tag Macrobrachium rosenbergii using implanted tags, and allows the tags to remain attached to the shrimp for extended periods, enabling the application of implanted tags in long-term production trials such as breeding.

[0056] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0057] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for individual marking of giant freshwater prawns, characterized in that, Includes the following steps: S1. Pre-marking preparation: Procurement of implantable glass tube encapsulated RFID chip tags, matching syringes or injection equipment, animal tissue adhesive, and thin plastic film discs. The shrimp to be tagged are temporarily kept until they reach a length of more than 3cm, which is from the base of the eyestalk to the end of the telson. S2. Preparation of shrimp to be tagged: Remove the shrimp to be tagged from the holding tank and place them in water at 16-22℃ to wait for tagged; S3, Tag Loading: The operator wears gloves and holds a label syringe to load the implantable glass tube-encapsulated RFID chip tag into the needle; S4. Adhesive for injection needle: After attaching the nozzle of the animal tissue adhesive, squeeze out a small amount of adhesive onto the syringe needle that has been labeled, so that the adhesive completely covers the needle hole for 2-3 mm. S5. Tag injection: Remove the shrimp to be labeled from the water, gently and evenly dry the shrimp body with an absorbent cloth, hold the cephalothorax and tail with one hand, and inject a glass tube label into the front half of the shrimp tail. S6. Needle hole injection: After the syringe needle is pulled out, seal the needle hole on the shrimp shell with animal tissue glue, making sure the glue covers the needle hole. S7. Pinhole sealing: Stick the prepared thin plastic film disc to the sealed pinhole, and gently rub the thin plastic film disc until it is completely and firmly stuck. S8. Settling gel: Settle the tagged shrimp in the gel, then return the tagged shrimp to the temporary container.

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