CN100544593C - A kind of seed selection has the method for the Environment of Litopenaeus vannamei Low parent shrimp of white spot syndrome virus resisting character - Google Patents
A kind of seed selection has the method for the Environment of Litopenaeus vannamei Low parent shrimp of white spot syndrome virus resisting character Download PDFInfo
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Abstract
The present invention relates to the method that a kind of seed selection has the Environment of Litopenaeus vannamei Low parent shrimp of white spot syndrome virus resisting character, its operation sequence is: 1) close shrimp is selected: select healthy close shrimp to carry out supporting temporarily according to male and female than 1: 2 ratio; 2) attack poison: the first attached joint intramuscular injection white spot syndrome virus liquid at close shrimp back; 3) eliminate through virus injection sequela and dead close shrimp, thereby filter out Environment of Litopenaeus vannamei Low parent shrimp with white spot syndrome virus resistance; 4) parent shrimp rearing: the close shrimp that will filter out is cultivated and fortification.The inventive method is simple, easy to operate, be suitable for the Environment of Litopenaeus vannamei Low parent shrimp that extensive seed selection has white spot syndrome virus resisting character.
Description
[technical field]
The present invention relates to the method that a kind of seed selection has the Environment of Litopenaeus vannamei Low parent shrimp of white spot syndrome virus resisting character.
[background technology]
Environment of Litopenaeus vannamei Low is the important economic shrimps of seawater, and characteristics such as, market value height strong because of its fast growth, resistance against diseases are subjected to aquaculturist's attention deeply, and breed scale in recent years is increasing.But along with the increase of culturing scale and the aggravation of water environment pollution, the disease in the Environment of Litopenaeus vannamei Low breeding process also happens occasionally, and wherein working the mischief maximum to breed is Environment of Litopenaeus vannamei Low white spot syndrome (white spotsyndrome is called for short WSS).Prawn white spot syndrome is to cause also do not have effective medicine to prevent and treat WSSV at present by white spot syndrome virus (white spotsyndrome virus, be called for short WSSV), therefore controls the main method that WSS breaks out and is prevention.It is found that aborning, there is the part prawn to have the ability of anti-WSSV in the Environment of Litopenaeus vannamei Low population, this part prawn can healthy survive after infecting WSSV, and filial generation also has antiviral characteristic, is called as SPR (special pathogen resistance) population.Therefore, the individuality that screening has an anti-WSSV ability in Environment of Litopenaeus vannamei Low parent shrimp is bred, and what utilize that the ntiviral characteristic of its seedling prevents prawn white spot syndrome is one of important measures that guarantee the sustainable development of Environment of Litopenaeus vannamei Low aquaculture.And at present, the screening technique with Environment of Litopenaeus vannamei Low parent shrimp of anti-WSSV ability is not also seen.
[summary of the invention]
The purpose of this invention is to provide the method that a kind of seed selection has the Environment of Litopenaeus vannamei Low parent shrimp of anti-WSSV characteristic.
The concrete steps of the Environment of Litopenaeus vannamei Low parent shrimp selection with anti-WSSV characteristic of the present invention are as follows:
1) close shrimp is selected and supports temporarily: according to the male and female ratio is that 1: 2 ratio is selected healthy close shrimp.The close shrimp body of described health length is that 14～18cm, body weight are 35～40g, build is big, body colour is normal, epipodite is complete, gill portion cleaning.The method for optimizing that described close shrimp is supported temporarily is: the density according to 30～50 tails/case is put into the PVC aquarium that chamber interior volume is 1000L with close shrimp, water in the aquarium is the processing of process ozonization, disinfection by ultraviolet light and aperture 1～5 μ m accurate filter earlier, carry out miniflow water according to 1L/ minute speed and change water, guarantee that during whole close shrimp seed selection water temperature is controlled at 26～30 ℃, salinity is 28～34, dissolved oxygen guarantees more than 5 mg/litre, PH7.8～8.6, total ammonia nitrogen content is lower than 0.6 mg/litre in the water.Every day throw something and feed 4 time according to 2% of the prawn body weight close shrimp feed (purchasing in Zhanjiang Evergreen Southern Ocean Technology Co., Ltd.) of throwing something and feeding every day.
2) attack poison: close shrimp is supported after 10 days in the 1000L aquarium temporarily, pulls prawn from aquarium fast out,, is put back in the aquarium then at the viral liquid 50 μ L of every tail parent shrimp back first attached joint intramuscular injection WSSV with disposable syringe.
Described WSSV virus liquid preferred manufacturing procedure is: the cultivating pool at the outburst white spot syndrome is taken a sample, and this shrimp pond white spot syndrome symptom is obvious, detects and throw something and feed to infect through PCR to prove the WSSV positive.Get the attached joint muscle of prawn of 0.1g outburst white spot syndrome and put into homogenizer, add the 1mLPBS buffer solution rapidly, ice bath grinds evenly, then viral liquid is inserted the centrifugal 5min of 3000r/min in the EP pipe of 1.5mL, get supernatant 200 μ L and join in the PBS buffer solution of 1800 μ L, use through what autoclave sterilization was crossed 0.22 μ L hole purpose filter filtration sterilization is housed.Get the PBS buffer solution mixing that filtrate 500 μ L join 49500 μ L (49.5mL) pH7.4, amount to 50mL, preserve the standby virus injection liquid of doing for 4 ℃.Get final product with 1000 times of uses of PBS buffer solution dilution before the injection.
3) eliminate non-resistance close shrimp: injecting virus liquid after the 2nd day prawn begin to occur infection symptoms, begin to occur dead after 3 days.Conscientiously observe that prawn infects and incidence every day, finding that dead shrimp is in time pulled out carries out harmless treatment and discards then; Attacking poison back, to culture after 8 days about 80% close shrimp morbidity dead, and remaining prawn does not take place to infect and disease symptom, illustrates that it has the ability of white spot syndrome virus resisting (WSSV).
4) parent shrimp rearing: the close shrimp that one-tenth survives is screened once more, selected body surface not have wounds, ingest well and the good close shrimp of vigor moves into indoor parent shrimp rearing pond and cultivates.The specification in parent shrimp rearing pond is generally 16～25 square metres, and 1～1.2 meter of the depth of water is put into close shrimp 80～200 tails in each cement pit; Cultivate pond water condition of water quality require with close shrimp temporarily the foster stage identical, quantity of exchanged water changes water 40%～70% every day.Periodicity of illumination is according to 12h: 12h control, and light intensity is controlled at 500～1000lx.With close shrimp manufactured feed (purchasing), sandworm, squid, beef liver etc. in Zhanjiang Evergreen Southern Ocean Technology Co., Ltd. according to 1: 3: 1: 1 the ratio close shrimp of feeding.The feeding volume of every day calculates according to 2% of close shrimp body weight, throws something and feeds every day 3～4 times.2～3 months breed phases, wait to culture close shrimp size when reaching 50～70g, can impel its gonadal maturation.Can carry out seed production routines such as mate and oviposit.
Breed base base, seed engineering south, national 863 ocean (Zhanjiang Evergreen Southern Ocean Technology Co., Ltd.) SPR seed shrimp in July, 2004, through 5000 tails parent shrimp is attacked the poison check, screening possesses Environment of Litopenaeus vannamei Low parent shrimp 1531 tails of anti-WSSV ability altogether, cultured through 3 months, survival rate 95%, mating rate 80%.Adopt the Environment of Litopenaeus vannamei Low parent shrimp of artificial culture, altogether production Environment of Litopenaeus vannamei Low nauplius tail more than 2.3 hundred million; Environment of Litopenaeus vannamei Low seed 6,900 ten thousand tails.The anti-WSSV Environment of Litopenaeus vannamei Low of first filial generation is cultured through commercial plant, can grow to 10～12g in 90 days, and all not have white spot syndrome to take place.In the laboratory, get the anti-WSSV Environment of Litopenaeus vannamei Low of a parton generation and carry out challenge test, found that its survival rate can reach 79%.2005, hybridized with other family prawns after the anti-WSSV prawn of a parton generation being cultivated become close shrimp again in this base, and the second filial of producing has better anti-virus ability and growth potential.
The present invention utilizes the principle of modern biology, by the individuality that has anti-WSSV ability in the stress test screening Environment of Litopenaeus vannamei Low parent shrimp.Breed its first filial generation through the close shrimp after this seed selection process, in two generations, all had the ability of stronger anti-WSSV, is fit to culture under various conditions, and the success rate that improves commercial prawn culturing is played an important role.
The invention provides the new method that a kind of healthy high quality seedling is bred, it is simple to operation, be suitable for the Environment of Litopenaeus vannamei Low parent shrimp that extensive seed selection has anti-WSSV characteristic.
[embodiment]
Embodiment 1:
1) close shrimp is selected and supports temporarily: long for 14cm, body weight are healthy close shrimp 5000 tails of 35g according to the ratio selective body of male and female than 1: 2, close shrimp build is big, body colour is normal, epipodite is complete, gill portion cleaning.Close shrimp is put into the PVC aquarium of indoor 1000L according to the density of 30 tails/case, water in the aquarium is that 1 μ m accurate filter is handled through ozonization, disinfection by ultraviolet light and aperture in advance, carry out miniflow water according to 1L/ minute speed then and change water, guarantee that during whole close shrimp seed selection water temperature is controlled at 26 ℃, salinity is 28, dissolved oxygen guarantees more than 5 mg/litre, PH7.8, total ammonia nitrogen content 0.6 mg/litre in the water.Every day throw something and feed 4 time according to 2% of the prawn body weight close shrimp feed (purchasing in Zhanjiang Evergreen Southern Ocean Technology Co., Ltd.) of throwing something and feeding every day.
2) attack poison: close shrimp is supported after 10 days in the 1000L aquarium temporarily, pulls prawn from aquarium fast out,, is put back in the aquarium then at the viral liquid 50 μ L of every tail parent shrimp back first attached joint intramuscular injection WSSV with disposable syringe.
Injection is used to such an extent that WSSV virus liquid is prepared as follows: the cultivating pool at the outburst white spot syndrome is taken a sample, and this shrimp pond white spot syndrome symptom is obvious, detects and throw something and feed to infect through PCR to prove the WSSV positive.Get the attached joint muscle of prawn of 0.1g outburst white spot syndrome and put into homogenizer, add the 1mLPBS buffer solution rapidly, ice bath grinds evenly, then viral liquid is inserted the centrifugal 5min of 3000r/min in the EP pipe of 1.5mL, get supernatant 200 μ L and join in the PBS buffer solution of 1800 μ L, use through what autoclave sterilization was crossed 0.22 μ L hole purpose filter filtration sterilization is housed.Get the PBS buffer solution mixing that filtrate 500 μ L join 49500 μ L (49.5mL) pH7.4, amount to 50mL, preserve the standby virus injection liquid of doing for 4 ℃.Can use after diluting 1000 times with PBS buffer solution before the injection.
3) eliminate non-resistance close shrimp: injecting virus liquid after the 2nd day prawn begin to occur infection symptoms, begin to occur dead after 3 days.Conscientiously observe that prawn infects and incidence every day, finding that dead shrimp is in time pulled out carries out discarding after the harmless treatment; Attacking poison back, to culture after 8 days about 80% close shrimp morbidity back dead, and remaining prawn does not infect, and does not see disease symptom, illustrates that it has the ability of white spot syndrome virus resisting (WSSV).
4) parent shrimp rearing: the close shrimp that one-tenth survives is screened once more, select body surface do not have wounds, ingest well and close shrimp that vigor is good totally 1531 tails move into indoor parent shrimp rearing pond and cultivate.The specification in parent shrimp rearing pond is 16 square metres, and 1.0 meters of the depth of waters are put into close shrimp 80 tails in each cement pit; Cultivate pond water condition of water quality require with close shrimp temporarily the foster stage identical, quantity of exchanged water changes water 40% every day.Periodicity of illumination is according to 12h: 12h control, and light intensity is controlled at 500x.With close shrimp manufactured feed (purchasing), sandworm, squid, beef liver etc. in Zhanjiang Evergreen Southern Ocean Technology Co., Ltd. according to 1: 3: 1: 1 the ratio close shrimp of feeding.The feeding volume of every day calculates according to 2% of close shrimp body weight, throws something and feeds every day 4 times.Culture after 2 months, close shrimp size reaches 60g, can carry out seed production routines such as mate and oviposit.
Embodiment 2:
1) close shrimp is selected and supports temporarily: long for 16cm, body weight are healthy close shrimp 2000 tails of 38g according to the ratio selective body of male and female than 1: 2, close shrimp build is big, body colour is normal, epipodite is complete, gill portion cleaning.Close shrimp is put into the PVC aquarium of indoor 1000L according to the density of 40 tails/case, water in the aquarium is the processing of 5 μ m accurate filters through ozonization, disinfection by ultraviolet light and aperture in advance, carry out miniflow water according to 1L/ minute speed and change water, guarantee that during whole close shrimp seed selection water temperature is controlled at 28 ℃, salinity is 30, dissolved oxygen guarantees 6 mg/litre, PH8.0, total ammonia nitrogen content 0.5 mg/litre in the water.Every day throw something and feed 4 time according to 2% of the prawn body weight close shrimp feed (purchasing in Zhanjiang Evergreen Southern Ocean Technology Co., Ltd.) of throwing something and feeding every day.
2) attack poison: close shrimp is supported after 10 days in the 1000L aquarium temporarily, pulls prawn from aquarium fast out,, is put back in the aquarium then at the viral liquid 50 μ L of every tail parent shrimp back first attached joint intramuscular injection WSSV with disposable syringe.
Injection is used to such an extent that WSSV virus liquid is prepared as follows: the cultivating pool at the outburst white spot syndrome is taken a sample, and this shrimp pond white spot syndrome symptom is obvious, detects and throw something and feed to infect through PCR to prove the WSSV positive.Get the attached joint muscle of prawn of 0.1g outburst white spot syndrome and put into homogenizer, add the 1mLPBS buffer solution rapidly, ice bath grinds evenly, then viral liquid is inserted the centrifugal 5min of 3000r/min in the EP pipe of 1.5mL, get supernatant 200 μ L and join 1800 μ LPBS buffer solutions, use through what autoclave sterilization was crossed 0.22 μ L hole purpose filter filtration sterilization is housed.Get the PBS buffer solution mixing that filtrate 500 μ L join 49500 μ L (49.5mL) pH7.4, amount to 50mL, preserve the standby virus injection liquid of doing for 4 ℃.Get final product with 1000 times of uses of PBS buffer solution dilution before the injection.
3) eliminate non-resistance close shrimp: injecting virus liquid after the 2nd day prawn begin to occur infection symptoms, begin to occur dead after 3 days.Conscientiously observe that prawn infects and incidence every day, finding that dead shrimp is in time pulled out carries out harmless treatment and discards then; Attacking poison back, to culture after 8 days about 80% close shrimp morbidity back dead, and remaining prawn does not infect, and does not see disease symptom, illustrates that it has the ability of white spot syndrome virus resisting (WSSV).
4) parent shrimp rearing: the close shrimp that one-tenth survives is screened once more, select body surface do not have wounds, ingest well and close shrimp that vigor is good totally 600 tails move into indoor parent shrimp rearing pond and cultivate.The specification in parent shrimp rearing pond is 25 square metres, and 1.2 meters of the depth of waters are put into close shrimp 150 tails in each cement pit; Cultivate pond water condition of water quality require with close shrimp temporarily the foster stage identical, quantity of exchanged water changes water 70% every day.Periodicity of illumination is according to 12h: 12h control, and light intensity is controlled at 800lx.With close shrimp manufactured feed (purchasing), sandworm, squid, beef liver etc. in Zhanjiang Evergreen Southern Ocean Technology Co., Ltd. according to 1: 3: 1: 1 the ratio close shrimp of feeding.The feeding volume of every day calculates according to 2% of close shrimp body weight, throws something and feeds every day 3 times.Culture after 3 months, close shrimp size reaches 50g, can carry out seed production routines such as mate and oviposit.
Embodiment 3:
1) close shrimp is selected and supports temporarily: long for 18cm, body weight are healthy close shrimp 4000 tails of 40g according to the ratio selective body of male and female than 1: 2, close shrimp selects to require that build is big, body colour is normal, epipodite is complete, gill portion cleans.Close shrimp is put into the PVC aquarium of indoor 1000L according to the density of 35 tails/case, water in the aquarium is the processing of 4 μ m accurate filters through ozonization, disinfection by ultraviolet light and aperture in advance, carry out miniflow water according to 1L/ minute speed and change water, guarantee that during whole close shrimp seed selection water temperature is controlled at 30 ℃, salinity is 34, dissolved oxygen guarantees 6 mg/litre, PH8.6, total ammonia nitrogen content 0.5 mg/litre in the water.Every day throw something and feed 4 time according to 2% of the prawn body weight close shrimp feed (purchasing in Zhanjiang Evergreen Southern Ocean Technology Co., Ltd.) of throwing something and feeding every day.
2) attack poison: close shrimp is supported after 10 days in the 1000L aquarium temporarily, pulls prawn from aquarium fast out,, is put back in the aquarium then at the viral liquid 50 μ L of every tail parent shrimp back first attached joint intramuscular injection WSSV with disposable syringe.
Injection is used to such an extent that WSSV virus liquid is prepared as follows: the cultivating pool at the outburst white spot syndrome is taken a sample, and this shrimp pond white spot syndrome symptom is obvious, detects and throw something and feed to infect through PCR to prove the WSSV positive.Get the attached joint muscle of prawn of 0.1g outburst white spot syndrome and put into homogenizer, add the 1mLPBS buffer solution rapidly, ice bath grinds evenly, then viral liquid is inserted the centrifugal 5min of 3000r/min in the EP pipe of 1.5mL, get supernatant 200 μ L and join in the PBS buffer solution of 1800 μ L, use through what autoclave sterilization was crossed 0.22 μ L hole purpose filter filtration sterilization is housed.Get the PBS buffer solution mixing that filtrate 500 μ L join 49500 μ L (49.5mL) pH7.4, amount to 50mL, preserve the standby virus injection liquid of doing for 4 ℃.Can use for 1000 times with the dilution of PBS buffer solution before the injection.
3) eliminate non-resistance close shrimp: injecting virus liquid after the 2nd day prawn begin to occur infection symptoms, begin to occur dead after 3 days.Conscientiously observe that prawn infects and incidence every day, finding that dead shrimp is in time pulled out carries out discarding after the harmless treatment; Attacking poison back, to culture after 8 days about 80% close shrimp morbidity back dead, and remaining prawn does not infect, and does not see disease symptom, illustrates that it has the ability of white spot syndrome virus resisting (WSSV).
4) parent shrimp rearing: the close shrimp that one-tenth survives is screened once more, select body surface do not have wounds, ingest well and close shrimp that vigor is good totally 1182 tails move into indoor parent shrimp rearing pond and cultivate.The specification in parent shrimp rearing pond is 25 square metres, and 1.2 meters of the depth of waters are put into close shrimp 200 tails in each cement pit; Cultivate pond water condition of water quality require with close shrimp temporarily the foster stage identical, quantity of exchanged water changes water 60% every day.Periodicity of illumination is according to 12h: 12h control, and light intensity is controlled at 1000lx.With close shrimp manufactured feed (purchasing), sandworm, squid, beef liver etc. in Zhanjiang Evergreen Southern Ocean Technology Co., Ltd. according to 1: 3: 1: 1 the ratio close shrimp of feeding.The feeding volume of every day calculates according to 2% of close shrimp body weight, throws something and feeds every day 4 times.Culture after 2 months, close shrimp size reaches 70g, can carry out seed production routines such as mate and oviposit.
Claims (2)
1, a kind of seed selection has the method for the Environment of Litopenaeus vannamei Low parent shrimp of white spot syndrome virus resisting character, it is characterized in that concrete steps are as follows:
1) close shrimp is selected: according to the ratio selective body length of male and female than 1: 2 is that 14～18cm, body weight are 35～40g, build is big, body colour is normal, epipodite is complete, the healthy close shrimp of gill portion cleaning carries out supporting temporarily; 2) attack poison: the first attached joint intramuscular injection white spot syndrome virus liquid 50 μ L at close shrimp back, wherein the preparation method of viral liquid is: the cultivating pool at the outburst white spot syndrome is taken a sample, this shrimp pond white spot syndrome symptom must obviously prove the white spot syndrome virus positive through the PCR detection and the infection of throwing something and feeding; Get the attached joint muscle of prawn of 0.1g outburst white spot syndrome then and put into homogenizer, add the 1mLPBS buffer solution rapidly, ice bath grinds evenly, then viral liquid is inserted the centrifugal 5min of 3000r/mi n in the EP pipe of 1.5mL, get the PBS buffer solution that supernatant 200 μ L join 1800 μ L, using through the aperture that autoclave sterilization is crossed is the filter filtration sterilization of 0.22 μ L; Then get the PBS buffer solution mixing that filtrate 500 μ L join 49500 μ L, pH7.4, preserve the standby virus injection liquid of doing for 4 ℃; Get viral liquid 1mL before the injection, can use after adding PBS buffer solution 999mL then; 3) eliminate through virus injection sequela and dead close shrimp, therefrom filter out Environment of Litopenaeus vannamei Low parent shrimp with white spot syndrome virus resistance; 4) parent shrimp rearing: the close shrimp that will screen is cultivated and fortification, the method of wherein cultivating the Environment of Litopenaeus vannamei Low parent shrimp with white spot syndrome virus resistance after screening is: the close shrimp that one-tenth survives is screened once more, select body surface not have wound, ingest good and the good close shrimp of vigor is put into 16～25 square metres, the parent shrimp rearing pond that the depth of water is 1～1.2 meter, density according to 80～200 tails/pond is cultivated, cultivating the pond water must be through ozonization, the processing of disinfection by ultraviolet light and 1～5 μ m accurate filter, every day, quantity of exchanged water was 40%～70%, periodicity of illumination is 12h: 12h, and light intensity is 500～1000lx; Then with close shrimp manufactured feed, sandworm, squid, beef liver etc. according to 1: 3: 1: 1 the ratio close shrimp of feeding, throw something and feed every day 3～4 times, a day feeding volume is 2% of a close shrimp body weight, 2～3 months breed phases, wait to culture close shrimp size when reaching 50～70g, can carry out the seed production routine of mate and oviposit.
2, selection as claimed in claim 1, the condition of supporting temporarily that it is characterized in that healthy close shrimp is: according to the density culture of 30～50 tails/1000L, water must be through the processing of ozonization, disinfection by ultraviolet light and 1～5 μ m accurate filter, carry out miniflow water according to 1L/ minute speed and change water, water temperature is 26～30 ℃, salinity is 28～34, dissolved oxygen is more than 5 mg/litre, PH7.8～8.6, total ammonia nitrogen content is 0～0.6 mg/litre in the water, throw something and feed every day close shrimp feed 4 times, a day feeding volume is 2% of a prawn body weight.
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| CNB2006101224240A CN100544593C (en) | 2006-09-26 | 2006-09-26 | A kind of seed selection has the method for the Environment of Litopenaeus vannamei Low parent shrimp of white spot syndrome virus resisting character |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102742523A (en) * | 2011-04-18 | 2012-10-24 | 中国水产科学研究院东海水产研究所 | Indoor cultivation method of litopenaeus vannamei parent |
| CN102613112B (en) * | 2012-03-12 | 2015-12-09 | 中国水产科学研究院东海水产研究所 | A kind of method for building up of prawn ' s virus infection model |
| CN102657109B (en) * | 2012-04-13 | 2013-12-25 | 中国水产科学研究院黄海水产研究所 | Equivalent and quantitative test method of white spot syndrome virus resistance of prawns |
| CN105918172A (en) * | 2016-04-29 | 2016-09-07 | 海南省海洋与渔业科学院 | Ecological culture method for Penaeus vannamei and Sipunculus nudus |
| CN113475440A (en) * | 2021-07-21 | 2021-10-08 | 新荣腾种业有限公司 | Method for breeding penaeus vannamei boone seedlings with glass seedling disease bacterium resistance |
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| 中国对虾抗病群体选育的初步研究. 张庆文，刘萍，王伟继，孔杰，王清印.海洋水产研究，第23卷第2期. 2002 |
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