CN114600811A - Method for purifying Chinese prawn core breeding group WSSV - Google Patents
Method for purifying Chinese prawn core breeding group WSSV Download PDFInfo
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- 241000238557 Decapoda Species 0.000 title claims abstract description 127
- 238000009395 breeding Methods 0.000 title claims abstract description 33
- 230000001488 breeding effect Effects 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 22
- 241000318927 Shrimp white spot syndrome virus Species 0.000 title claims 6
- 238000001514 detection method Methods 0.000 claims abstract description 47
- 238000005070 sampling Methods 0.000 claims abstract description 17
- 238000003306 harvesting Methods 0.000 claims abstract description 3
- 210000003608 fece Anatomy 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 4
- 235000013601 eggs Nutrition 0.000 claims description 3
- 210000000514 hepatopancreas Anatomy 0.000 claims description 3
- 238000007857 nested PCR Methods 0.000 claims description 3
- 241000696962 White spot syndrome virus Species 0.000 abstract description 40
- 238000000746 purification Methods 0.000 abstract description 8
- 230000012447 hatching Effects 0.000 abstract 1
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000008030 elimination Effects 0.000 description 5
- 238000003379 elimination reaction Methods 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
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- 238000009360 aquaculture Methods 0.000 description 4
- 244000144974 aquaculture Species 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 241000995704 Fenneropenaeus chinensis Species 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 230000005571 horizontal transmission Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
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- 102000002322 Egg Proteins Human genes 0.000 description 1
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- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
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- A—HUMAN NECESSITIES
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- A01K61/00—Culture of aquatic animals
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Abstract
The invention discloses a method for purifying a core breeding group WSSV of Chinese prawn, which comprises the following steps: collecting the excrement of the parent shrimps of the Chinese prawns in the overwintering pond to carry out WSSV detection, and eliminating the parent shrimps in the overwintering pond with positive detection results; carrying out single detection on the selected and remained parent shrimps, and eliminating individuals with positive detection results; selecting parent shrimps for spawning and hatching, and then sampling, mixing and detecting the shrimps in the nursery pond to eliminate positive shrimps; placing the shrimp individuals with negative detection results in the enclosure of the outdoor culture pond for culture; and sampling and mixing the cultured individuals in each enclosure during harvesting, selecting and reserving copulated female shrimps with negative detection results in the enclosure for overwintering, and taking the copulated female shrimps as parent shrimps bred in the second-year seedling. By adopting the purification scheme to continuously purify the core breeding population of the Chinese prawn for multiple generations, the risk of spread of WSSV (white spot syndrome Virus) can be obviously reduced, and the breeding success rate and the product quality safety of the Chinese prawn are improved.
Description
Technical Field
The invention belongs to the technical field of Chinese prawn breeding, and particularly relates to a method for purifying a WSSV (white spot syndrome virus) of a Chinese prawn core breeding group.
Background
Chinese prawn (Fenneropenaeus chinensis), also known as oriental prawn and Chinese prawn, is a unique species in offshore locality in China. Chinese prawn meat is fresh, tender and delicious, has high nutritive value, is an important breeding species in coastal areas such as Jiangsu, Shandong, Hebei and Liaoning in China all the time, and has the breeding yield of about 20 ten thousand tons in the prime period of breeding development (before and after 1990), which accounts for 70 percent of the breeding yield of Chinese prawn. In 1993, due to the outbreak of large-scale White Spot Syndrome Virus (WSSV), the culture yield of Chinese shrimps is sharply reduced, the annual yield is about 5 ten thousand tons for a long time, and economic losses are disastrous.
The WSSV has the characteristics of wide host range, strong infection capacity, high mortality and the like, the death rate of the Chinese prawn can reach 100 percent after the Chinese prawn is infected with the WSSV in 3 to 10 days, and the propagation mode can be horizontal propagation in a culture water body or vertical propagation from parent to offspring. In order to reduce the loss of WSSV to aquaculture industry, researchers have conducted a great deal of research on the interaction mechanism between the WSSV genome and the host, the improvement of detection methods, the prevention and treatment of the WSSV genome, the detection methods and the like, but no effective preventive vaccine and therapeutic drug exist at present. Therefore, the method has the advantages that purification measures are taken for core breeding groups of Chinese prawns, the detection of parent prawns and fries is enhanced, the probability of virus carrying of the fries of the prawns is reduced from the source, and the method has important significance for guaranteeing the quality of germplasm resources of the Chinese prawns and preventing the spread and the popularity of WSSV.
The aquaculture industry is the basis and the source of the aquaculture industry, and if the seedlings of the core breeding group carry pathogens, the whole aquaculture industry is bound to face serious epidemic disease risks.
Disclosure of Invention
The invention provides a method for purifying a core breeding group WSSV of Chinese prawns, which is characterized in that on the basis of fine breed breeding of the Chinese prawns, the method focuses on the White Spot Syndrome Virus (WSSV) which has the most serious harm to the breeding industry of the Chinese prawns, comprehensively reduces the risk of spread of the WSSV in the core breeding group of the Chinese prawns by comprehensively monitoring and continuously purifying parent prawns, offspring seeds and the WSSV in the breeding process, promotes the change from effective control to gradual purification and elimination of the disease control of the Chinese prawns, and improves the breeding success rate and the product quality safety of the Chinese prawns.
The invention provides a method for purifying a core breeding group WSSV of Chinese prawn, which comprises the following operation steps:
(1) collecting feces of copulated female shrimps (parent shrimps) of Chinese prawns in the overwintering pool, carrying out WSSV detection on the feces of the parent shrimps by a nested PCR detection method, and eliminating the parent shrimps in the overwintering pool with positive detection results; the number of the parent shrimps in each overwintering pond is controlled to be about 200, 3 parts of the parent shrimp feces are collected respectively at three time points of morning, noon and evening and are respectively put into sterilized centrifugal tubes of 1.5mL, and the parent shrimps are continuously collected for 3 days. The price of each parent shrimp is very high, the number of each overwintering pond is controlled to be 200, if the excrement is detected to be positive, the parent shrimps in the overwintering pond are eliminated, and the large economic loss is avoided; in addition, excrement is collected for 3 consecutive days to be sampled, so that most of parent shrimps can be sampled almost, and detection burden cannot be caused.
(2) Carrying out single detection on the selected and remained parent shrimps, and eliminating individuals with positive detection results; the step ensures that each parent shrimp selected and reserved does not carry WSSV, and simultaneously, because the quantity of the core breeding group is controlled to be about 1000, the detection of each parent shrimp does not have too much workload; if the step (1) is not used for screening, the detection workload of the step is large, and in actual production, the positive rate of overwintering parent shrimps is over 50 percent, so that at least parent shrimps over 2000 can be detected if 1000 parents which do not carry WSSV are screened.
(3) The selected parent shrimps are laid eggs and incubated, when the larvae grow to the larvae setting specification (the body length is about 1.0cm), the larvae in each nursery pond are sampled, mixed and detected, and positive larvae are eliminated;
(4) placing the shrimp individuals with negative detection results in an outdoor culture pond for culture; the selected and remained young shrimp individuals are cultured in a lattice surrounding mode, and the culturing quantity of each lattice surrounding is controlled to be 1000-; the cultivation in the separate enclosures can prevent a large amount of infection, and once a positive enclosure is found, individuals cultivated in the enclosure are eliminated, so that large-area propagation cannot be caused;
(5) sampling and mixing the cultured individuals in each enclosure during harvesting, selecting and reserving copulated female shrimps with negative detection results in the enclosure for overwintering, and using the copulated female shrimps as parent shrimps bred in the second-year seedling;
(6) repeating the steps (1) - (5) and continuously purifying for more than 5 generations.
The further technical scheme is as follows:
and (3) numbering the parent shrimps by adopting an eyestalk marking method in the step (2), wherein the sampling tissue is the second pair of footsteps of the Chinese shrimps.
In the step (3), 3 mixed sampling samples are sampled in each nursery pond, the number of the shrimps in each sample is 30-50, the sampling tissue is full shrimps, and the sampling is continuously performed for 3 days.
In the step (5), 3 samples are taken for mixed sampling of each enclosure, the number of prawns in each sample is 10, and the sampling tissue is the hepatopancreas.
Compared with the prior art, the invention has the beneficial effects that:
under the condition of WSSV flooding, the infection rate of Chinese prawn culture is about 40%, some individuals are infected and infected with the disease, some individuals carry viruses but do not develop the disease, and the potential infection sources can infect the WSSV to the same generation of individuals through horizontal transmission and can also infect the next generation through environment, sperm or ovum vertical transmission; how to cut off the infection source and establish a nontoxic core breeding parent is the basis for obtaining healthy filial generation, the method of the invention strictly selects from the parent through a closed loop process management method, and the risk of carrying WSSV is reduced in each step. The risk of WSSV horizontal transmission and vertical transmission is controlled from the source, so that healthy shrimps without viruses can be obtained, and the risk of diseases in the culture process is reduced.
Drawings
FIG. 1 is a flow chart of WSSV purification technology of the Chinese prawn core breeding group.
Detailed Description
The present invention will be described in detail with reference to the following embodiments in order to make the features and advantages of the invention more comprehensible. This invention may, however, be embodied in many different forms than those specifically described herein, and it will be apparent to those skilled in the art that many more modifications are possible without departing from the spirit and scope of the invention. The specific flow chart is shown in fig. 1.
Example 1: WSSV (white Spotted SV) detection and elimination of dejection of parent shrimps in core breeding group of Chinese shrimps in overwintering pond
In 2017, in 1 month, excrement of 24 parent shrimps in the overwintering pond of Chinese prawns is collected, the number of the parent shrimps in each overwintering pond is about 200, the parent shrimps are collected respectively at three time points of morning, noon and evening, the collection is continuously carried out for 3 days, the collected samples are respectively put into a sterilized 1.5mL centrifuge tube and are stored at the temperature of-20 ℃ for standby, and each sample is about 1-5 g. The WSSV detection is carried out on 216 collected samples, and the detection method is carried out according to the part 2 of the national standard' white spot syndrome (WSD) diagnosis procedure drafted by the yellow sea aquatic product research institute of the Chinese aquatic product science institute: nested PCR assay (GB/T28630.2-2012) "was performed. According to the WSSV detection result in the feces of the parent shrimps, 16 overwintering ponds with positive detection results of eliminated samples are selected, and the positive detection rate is 66.7%. The selected 1500-tailed parent shrimps are separately cultured in an isolated way, and the used equipment comprises indoor overwintering pool walls, air stones, hoses, filter bolting silk and the like, which are thoroughly disinfected and managed by special people.
Example 2: WSSV detection and elimination of individual parent shrimps
Before the selected and remained parent shrimps are transferred from the overwintering pond to the spawning pond, each selected and remained parent shrimp is subjected to WSSV detection, the detected parent shrimps are distinguished by eye marks marked with numerical numbers, the sampling tissue is the second pair of feet of the Chinese shrimps, and the Chinese shrimps are placed into a sterilized 1.5mL centrifuge tube and are stored for later use at the temperature of minus 20 ℃. And (3) carrying out WSSV detection on 1360 parent shrimps in total, and eliminating 243 tails of positive parent shrimps, wherein the positive detection rate is 17.9%. And (4) transferring the selected and remained parent shrimps into a sterilized spawning pond to spawn and hatch.
Example 3: WSSV (Wireless sensor System) detection and elimination of juvenile shrimps of Chinese prawns
After the parent shrimps are selected and kept to lay eggs and hatch, the parent shrimps are selected and keptThe collected nauplii are put into 30 water bodies of 1m3The larva breeding pond is used for culturing the larva, and the number of the larva in each pond is controlled to be about 10 thousands of tails. The apparatus, fresh and live bait, water quality and the like used in the process of larva cultivation are regularly disinfected. And when the larvae grow to the larvae release specification (the body length is about 1cm), randomly fishing the larvae from each nursery pond to detect the WSSV. Randomly sampling 3 samples in each nursery pond, controlling the number of the shrimps in each sample to be 30-50, continuously sampling for 3 days, eliminating 12 nursery ponds with positive detection results, and obtaining the sample positive detection rate of 40%.
Example 4: breeding of Chinese prawn selecting and remaining young prawn
From a nursery pond with negative sample detection, selecting robust and high-activity shrimps to be placed in an outdoor culture pond, and culturing in a lattice manner, wherein the number of the seedlings placed in each lattice is about 1000, and the number of culture lattices is 60. And implementing healthy cultivation management measures in the cultivation process.
Example 5: backup parent shrimp WSSV detection and elimination
When the penaeus chinensis is harvested in 11 months in 2017, respectively randomly taking 30 penaeus chinensis from each enclosure for WSSV detection, setting 3 parallel samples in total, wherein the number of each sample is 10, and putting the hepatopancreas tissues into a sterilized 1.5mL centrifuge tube for storage at-20 ℃ for later use. 26 grids with positive detection results of eliminated samples are provided, and the positive detection rate of the samples is 43.3%. Selecting backup parent shrimps with strong constitution and large individuals from the surrounding lattices with negative detection results, putting the backup parent shrimps with 5000 tails into an indoor culture pond for overwintering, and reserving the backup parent shrimps as parent shrimps bred in the second-year seedling.
Example 6: purification effect of continuous 5 generations of Chinese prawn core breeding group
In 2017 and 2021, the core breeding population of the Chinese prawns is continuously purified for 5 generations according to the purification process, and the purification effect is shown in the following table. As can be seen from the table, the WSSV positive detection rate of each breeding generation of the Chinese shrimps is in a significantly reduced trend, the positive detection rates of the excrements of the parent shrimps, the individual parent shrimps, the shrimps and the backup parent shrimps are respectively reduced from 66.7%, 17.9%, 40.0% and 43.3% of the F1 generation to 31.8%, 9.8%, 19.4% and 21.8% of the F5 generation, and the purification effect is significant.
All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (4)
1. A method for purifying a core breeding group WSSV of Chinese prawns is characterized by comprising the following operation steps:
(1) collecting the feces of the copulated female shrimps of the Chinese prawns in the overwintering pool, carrying out WSSV detection on the feces of the parent shrimps by a nested PCR detection method, and eliminating the parent shrimps in the overwintering pool with positive detection results; the number of the parent shrimps in each overwintering pond is controlled at 180-200, and the excrement of the parent shrimps is collected at three time points of morning, noon and evening respectively and is continuously collected for 3 days;
(2) carrying out single detection on the selected and remained parent shrimps, and eliminating individuals with positive detection results;
(3) the selected parent shrimps are laid eggs and incubated, and when the larvae grow to the larvae release specification, the larvae in each nursery pond are sampled, mixed and detected, so that positive larvae are eliminated;
(4) placing the shrimp individuals with negative detection results in an outdoor culture pond for culture; the selected and remained young shrimp individuals are cultured in a lattice surrounding mode, and the culturing quantity of each lattice surrounding is controlled to be 1000-;
(5) sampling and mixing the cultured individuals in each enclosure during harvesting, selecting and reserving copulated female shrimps with negative detection results in the enclosure for overwintering, and using the copulated female shrimps as parent shrimps bred in the second-year seedling;
(6) repeating the steps (1) - (5) and continuously purifying for more than 5 generations.
2. The method for purifying the WSSV of the core breeding group of the Chinese shrimps as claimed in claim 1, wherein the eyestalk marking method is adopted in the step (2) to number the parent shrimps, and the sampling tissue is the second pair of feet of the Chinese shrimps.
3. The method for purifying the WSSV of the core breeding group of Chinese shrimps as claimed in claim 1, wherein in the step (3), the number of the mixed sampling samples in each nursery pond is 3, the number of the shrimps in each sample is 30-50, the sampling tissue is the whole shrimps, and the sampling is performed continuously for 3 days.
4. The method for purifying the WSSV of the core breeding group of Chinese shrimps as claimed in claim 1, wherein the number of the samples tested in step (5) is 3 for each enclosure, the number of the shrimps in each sample is 10, and the sampled tissue is the hepatopancreas.
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| CN1367834A (en) * | 1999-08-03 | 2002-09-04 | 阿克佐诺贝尔公司 | Proteins derived from white spot syndrome virus and uses thereof |
| CN1745725A (en) * | 2005-09-13 | 2006-03-15 | 中国水产科学研究院南海水产研究所 | Artificial culture of shrimp parents |
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| CN108342512A (en) * | 2018-05-09 | 2018-07-31 | 上海海洋大学 | A method of quantitatively detecting White Spot Syndrome Virus using TaqMan probe |
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| CN114271225A (en) * | 2020-12-31 | 2022-04-05 | 渤海水产股份有限公司 | Method for effectively preventing and treating white spot syndrome in salt pan shrimp culture |
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- 2022-04-21 CN CN202210421679.6A patent/CN114600811A/en active Pending
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| CN1367834A (en) * | 1999-08-03 | 2002-09-04 | 阿克佐诺贝尔公司 | Proteins derived from white spot syndrome virus and uses thereof |
| US20090068212A1 (en) * | 1999-08-03 | 2009-03-12 | Intervet International B.V. | Proteins derived from white spot syndrome virus and uses thereof |
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| CN108342512A (en) * | 2018-05-09 | 2018-07-31 | 上海海洋大学 | A method of quantitatively detecting White Spot Syndrome Virus using TaqMan probe |
| CN110679524A (en) * | 2019-09-19 | 2020-01-14 | 海南海壹水产种苗有限公司 | Indoor industrialized ecological specific pathogen-free seedling cultivation method for litopenaeus vannamei |
| CN114271225A (en) * | 2020-12-31 | 2022-04-05 | 渤海水产股份有限公司 | Method for effectively preventing and treating white spot syndrome in salt pan shrimp culture |
Non-Patent Citations (1)
| Title |
|---|
| 厦门市海洋与渔业局: ""对虾无公害健康养殖技术"", 中国农业出版社, pages: 125 - 173 * |
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