CN104255586B - A kind of time point quantitative Treatment method of producing Hong Kong oyster all-triploid - Google Patents
A kind of time point quantitative Treatment method of producing Hong Kong oyster all-triploid Download PDFInfo
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Abstract
The invention discloses a kind of time point quantitative Treatment method of producing Hong Kong oyster all-triploid.The present invention, by obtaining monomer essence ovum, process fertilized egg, detect the sport technique segments such as larva ploidy in good time, creates a kind of time point quantitative Treatment method using the development of fertilized ova stage as biological indicator, can obtain 100% Hong Kong oyster triploid.The present invention adopt monomer essence ovum preferably, pairing, effectively eliminate the defect mixing smart ovum poor synchronization in oyster triploid Induction Process in the past, the zygote that the synchronism of acquisition is higher.The present invention has overturned and has occurred that first polar body is as processing time point using 30-50% traditionally, and process 20min is the fixing thoughtcast of Best Times section; But using the development of fertilized ova stage as biological indicator, adopt A+1/3B as processing time point, B+C, as processing time section, has avoided the impact that the external environmental conditions such as temperature process it, successfully can induce Hong Kong oyster all-triploid.
Description
Technical field:
The invention belongs to shellfish Biotechnology in Genetic Breeding field in Marine agriculture, be specifically related to a kind of time point quantitative Treatment method of producing Hong Kong oyster all-triploid.
Background technology:
China is oyster culture big country, and 09-13 annual production is all 350-380 about ten thousand tons; And the output of Hong Kong oyster (Crassostreahongkongensis) is at about 1,300,000 tons, it accounts for 30% of oyster output, 12% of cultivated shellfish output, 7% of mariculture gross yield, more than 21% (China Fisheries yearbook, 2014) of world wide production.Hong Kong oyster is distributed in Guangdong of China, Guangxi, Fujian, it is the shellfish that a kind of economic worth of warm warm nature offshore growth is very high, hobby high-temperature low salt environment, its meat flavour is delicious, be rich in protein and multiple essential amino acid thereof, being described as " milk in ocean ", is a kind of important living marine resources.It has the individual feature such as large, nutritious, deeply favors by consumers in general; Except eating, be processed into that Ho is dry raw, except Ho fermented soya beans, salted or other wise, or the important source material of the South China coastal oyster deep processing high-end product (oyster high level extract, oyster health-caring capsule, oyster calcium etc.).
Cytochalasin B (CB) is adopted to process American oyster (C.virginica) fertilized egg the earliest from (1980) such as Stanley, since obtaining triploid filial generation in 8 months, shellfish triploid Breeding technology attention.The growth of triploid shellfish tool is fast, individual large, fertility is poor, quality better, the lethality mating season advantage such as low.Afterwards in long oyster (C.gigas) (Allen etc., 1986; Guo etc., 1996; Yu Ruihai etc., 2000; Wang Zhaoping etc., 2004; Hole is waited quietly, 2011), edible oyster (Ostreaedulis) (Gendreau etc., 1990), Sydney rock oyster (Saccostreacommercialis) (Nell, 1995) and Portuguese oyster (C.angulata) (Zeng Zhinan etc., 1994) etc. also in succession successfully induce triploid in oyster, but almost there is not yet the relevant report of Hong Kong oyster polyploid breeding.
The breeding of oyster multiploid induction has developed more than 30 year, and scholars' successful incubation goes out long oyster tetraploid, obtains all-triploid (Piferrer etc., 2009) by tetraploid and diploid hybrid.But for not yet obtaining for tetraploid oyster species, direct induced triploid remains main polyploid production method, and it is not high that traditional abductive approach still also exists times rate, and can operational stability poor wait not enough.
Summary of the invention:
The object of the invention is to solve the inductivity existed in traditional triploid preparation method lower, and the defect such as doubly rate is unstable, thus propose a kind of time point quantitative Treatment method using the development of fertilized ova stage as biological indicator, 100% Hong Kong oyster triploid can be produced.The present invention has the advantages such as easy and simple to handle, practical, efficiently easy, and this cultivates for Hong Kong oyster triploid and solid theory and practice basis has been established in tetraploid production.
The time point quantitative analysis method of production Hong Kong of the present invention oyster all-triploid, is characterized in that, comprise the following steps:
A, obtain monomer ovum: Hong Kong oyster mating season (the 4-8 month), using same geographical population as core population for material, choose ripe 3-5 Hong Kong in age oyster female individuals; Select with ovum quality and quantity dual indexes, pick out and grow neatly, ten thousand, ovum quantity >=5000, egg membrane is complete, and yolk accumulation is rendered as the individuality of buff as female parent; Collecting monomer ovum, is soak 30-60min in 15-20ppt seawater in salinity, after its germinal vesicle breakdown is complete, then is 15-20ppt seawater cleaning ovum by fresh salinity, obtains monomer ovum liquid.Ovum recycles fresh seawater cleaning 2-3 time, and its form is circular or oval;
B, acquisition monomer sperm: with the core population in a step for material, filter out the male in 2-3 age; Afterwards its sperm quality is detected, under filtering out microscope >=individuality of 30% sperm motility, in salinity 15-20ppt seawater, activate 10-15min; Microscopy again afterwards, using the individuality of >=90% sperm motility as male parent, collects monomer sperm;
C, in good time process fertilized egg: monomer sperm is added in monomer ovum liquid, control each ovum periphery and have 3-8 sperm, ovum density domination is at≤5 ten thousand/mL, during this period, temperature controls at 25-32 DEG C, salinity controls at 15-24ppt, wherein, from sperm adds, to finding that the time period that 10% first polar body is released is set to A, the time period of releasing from first polar body to discovery 10% second polar body is set to B, time period from second polar body to discovery 10% spilting of an egg individuality is set to C, select to adopt conventional method process fertilized egg at (A+1/3B) time point, suppress the discharge of fertilized egg second polar body, the process duration is (B+C), be positioned in normal seawater after complete and hatched, namely Hong Kong oyster triploid D shape larva can be obtained.
Detect larva ploidy: carrying out Ploidy detection for obtaining first D shape larva employing flow cytometer of incubating in step c, finding that all larvas are all in triploid state, producing without dliploid and aneuploid.
Its fertilized egg can normal incubation, all-triploid filial generation can be obtained according to the conventional breeding method of these species.
Described conventional method process fertilized egg refers to and utilizes cytochalasin B (CB) or 6-dimethylaminopurine process fertilized egg, does not comprise the physical methods such as hypotonic, cold and hot temperature shock and hydrostatic pressing.
The direct abductive approach of the different traditional oyster triploids of the present invention, also exists following main innovate point: 1. pairing strategy is different: the mixing essence ovum that conventional method adopts is as induction object, and zygote developing synchrotron is poor; And the present invention is by the pairing of monomer essence ovum, zygote synchronism is very high, is close to consistent.2. opportunity is processed different: conventional process time point occurs first polar body in 30-50% fertilized egg, and the present invention is using the development of fertilized ova stage as biological indicator, occur being designated as time period A to 10% first polar body from fertilization, occur being designated as time period B from first polar body to appearance 10% second polar body, be released into appearance 10% spilting of an egg zygote from second polar body and be designated as time period C, discharge complete at fertilized egg first polar body 100%, namely process and control in A+1/3B best results opportunity.3. process duration determination methods different: the conventional process duration is fixed as 20min, makes it occur changing by a relatively large margin the triploid of varying environment, and doubly rate is unstable; And the present invention is using development of fertilized ova as biological indicator, using the B+C time period as the process duration, be generally 15-30min, effectively avoided the impact that the envirment factor such as temperature, salinity is grown it, ensure that second polar body is effectively suppressed in zygote, thus form all-triploid.4. result is obtained different: conventional method induced triploid rate changes at 60-90%, often occurs aneuploid; And triploid rate of the present invention is stabilized in 100%, and occur without dliploid, tetraploid and aneuploid.
The present invention, by obtaining monomer essence ovum, process fertilized egg, detect the sport technique segments such as larva ploidy in good time, creates a kind of time point quantitative Treatment method using the development of fertilized ova stage as biological indicator, can obtain 100% Hong Kong oyster triploid.The present invention adopt monomer essence ovum preferably, pairing, effectively eliminate the defect mixing smart ovum poor synchronization in oyster triploid Induction Process in the past, the zygote that the synchronism of acquisition is higher.The present invention has overturned and has occurred that first polar body is as processing time point using 30-50% traditionally, and process 20min is the fixing thoughtcast of Best Times section; But using the development of fertilized ova stage as biological indicator, adopt A+1/3B as processing time point (100% first polar body release is complete), B+C is as processing time section (15-30min), avoid the impact that the external environmental conditions such as temperature process it, successfully can induce Hong Kong oyster all-triploid.The present invention is that Hong Kong oyster polyploid breeding has established solid theory and practice basis.The advantages such as the present invention has easy and simple to handle, practical, efficiently easy.
Accompanying drawing illustrates:
Fig. 1 is schematic diagram of the present invention.The first half is conventional method, and the latter half is the technology of the present invention route map.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1
A, acquisition monomer ovum: in late June, 2013, Hong Kong oyster in experiment centre this locality, Chinese Academy of Science Nanhai Ocean Research Institute Zhanjiang, as material, chooses ripe 3-5 Hong Kong in age oyster female individuals 30; Select with ovum quality and quantity dual indexes, pick out and grow neatly, ovum quantity average out to 8,000 ten thousand, egg membrane is complete, and yolk accumulation is rendered as the individuality of buff as female parent 6; Collect monomer ovum, be soak 45min in 18ppt seawater in salinity, treat that its germ-vesicle 100% breaks completely, obtain ovum.The fresh seawater that ovum utilizes salinity to be 18ppt cleans 3 times repeatedly, and obtain monomer ovum liquid, ovum is circle or ellipse.
B, acquisition monomer sperm: with the core population in a step for material, filter out the male several 12 in 2-3 age; Afterwards its sperm quality is detected, under filtering out microscope >=individuality of 30% sperm motility, in salinity 18ppt seawater, activate 12min; Microscopy again afterwards, using 6 of >=90% sperm motility individualities as male parent, obtains monomer whose sperm.
C, process fertilized egg: added by 6 monomer sperms in 6 monomer ovum liquid, have 3-8 sperm to be advisable with each ovum periphery, ovum density is≤5 ten thousand/mL in good time.During this period, temperature is 29.6 DEG C, and salinity is 18ppt.Wherein, from sperm adds, to finding that the time period that 10% first polar body is released is set to A (10min), the time period of releasing from first polar body to discovery 10% second polar body is set to B (12min), and the time period from second polar body to discovery 10% spilting of an egg individuality is set to C (9min).Select at (A+1/3B) time point, namely after fertilization 14min utilizes 0.5mg/L cytochalasin B (CB) to process fertilized egg, suppresses the discharge of fertilized egg second polar body; The process duration is (B+C), is positioned in normal seawater and hatches, namely can obtain 100% Hong Kong oyster triploid D shape larva after namely processing 21min.
D, detection larva ploidy: carrying out Ploidy detection for obtaining first D shape larva employing flow cytometer of incubating in step c, finding that all larvas are all in triploid state, producing without dliploid and aneuploid.After this, all-triploid filial generation is raised according to the conventional breeding method of these species.
Embodiment 2
A, acquisition monomer ovum: in late May, 2014, at Chinese Academy of Science Nanhai Ocean Research Institute's Zhanjiang experiment centre using Hong Kong oyster of Zhuhai colony as material, choose ripe 3-5 Hong Kong in age oyster female individuals 25; Select with ovum quality and quantity dual indexes, pick out and grow neatly, ovum quantity average out to 12,000 ten thousand, egg membrane is complete, and yolk accumulation is rendered as the individuality of buff as female parent 5; Collect monomer ovum, be soak 30min in 20ppt seawater in salinity, treat that its germ-vesicle 100% breaks completely, obtain ovum.The fresh seawater that ovum utilizes salinity to be 20ppt cleans 3 times repeatedly, and obtain monomer ovum liquid, ovum is circle or ellipse.
B, acquisition monomer sperm: with the core population in a step for material, filter out the male several 10 in 2-3 age; Afterwards its sperm quality is detected, under filtering out microscope >=individuality of 30% sperm motility, in salinity 20ppt seawater, activate 15min; Microscopy again afterwards, using 5 of >=90% sperm motility individualities as male parent, obtains monomer whose sperm.
C, process fertilized egg: added by 5 monomer sperms in 5 monomer ovum liquid, have 3-8 sperm to be advisable with each ovum periphery, ovum density is≤5 ten thousand/mL in good time.During this period, temperature is 27.5 DEG C, and salinity is 20ppt.Wherein, from sperm adds, to finding that the time period that 10% first polar body is released is set to A (12min), the time period of releasing from first polar body to discovery 10% second polar body is set to B (15min), and the time period from second polar body to discovery 10% spilting of an egg individuality is set to C (10min).Select at (A+1/3B) time point, namely after fertilization 17min utilizes 0.5mg/L cytochalasin B process fertilized egg, suppresses the discharge of fertilized egg second polar body; The process duration is (B+C), is positioned in normal seawater and hatches, namely can obtain 100% Hong Kong oyster triploid D shape larva after namely processing 25min.
D, detection larva ploidy: carrying out Ploidy detection for obtaining first D shape larva employing flow cytometer of incubating in step c, finding that all larvas are all in triploid state, producing without dliploid and aneuploid.After this, all-triploid filial generation is raised according to the conventional breeding method of these species.
Embodiment 3
A, acquisition monomer ovum: the first tenday period of a month in May, 2014, at shellfish complex experiment station, Guangxi using Hong Kong oyster of Mao Weihai colony as material, choose ripe 3-5 Hong Kong in age oyster female individuals 27; Select with ovum quality and quantity dual indexes, pick out and grow neatly, ovum quantity average out to 6,000 ten thousand, egg membrane is complete, and yolk accumulation is rendered as the individuality of buff as female parent 9; Collect monomer ovum, be soak 40min in 16ppt seawater in salinity, treat that its germ-vesicle 100% breaks completely, obtain ovum.The fresh seawater that ovum utilizes salinity to be 16ppt cleans 3 times repeatedly, and ovum is circle or ellipse.
B, acquisition monomer sperm: with the core population in a step for material, filter out the male several 15 in 2-3 age; Afterwards its sperm quality is detected, under filtering out microscope >=individuality of 30% sperm motility, in salinity 16ppt seawater, activate 10min; Microscopy again afterwards, using 9 of >=90% sperm motility individualities as male parent, obtains monomer sperm.
C, process fertilized egg: added by 9 monomer sperms in 9 monomer ovum liquid respectively, have 3-8 sperm to be advisable with each ovum periphery, ovum density is≤5 ten thousand/mL in good time.During this period, temperature is 25.4 DEG C, and salinity is 16ppt.Wherein, from sperm adds, to finding that the time period that 10% first polar body is released is set to A (14min), the time period of releasing from first polar body to discovery 10% second polar body is set to B (18min), and the time period from second polar body to discovery 10% spilting of an egg individuality is set to C (12min).Select at (A+1/3B) time point, namely after fertilization 20min utilizes 0.5mg/L cytochalasin B process fertilized egg, suppresses the discharge of fertilized egg second polar body; The process duration is (B+C), is positioned in normal seawater and hatches, namely can obtain 100% Hong Kong oyster triploid D shape larva after namely processing 30min.
D, detection larva ploidy: carrying out Ploidy detection for obtaining first D shape larva employing flow cytometer of incubating in step c, finding that all larvas are all in triploid state, producing without dliploid and aneuploid.After this, all-triploid filial generation is raised according to the conventional breeding method of these species.
Embodiment 4
A, acquisition monomer ovum: in early July, 2014, at shellfish complex experiment station, Guangxi using Hong Kong oyster of Mao Weihai colony as material, choose ripe 3-5 Hong Kong in age oyster female individuals 21; Select with ovum quality and quantity dual indexes, pick out and grow neatly, ovum quantity average out to 5,000 ten thousand, egg membrane is complete, and yolk accumulation is rendered as the individuality of buff as female parent 4; Collect monomer ovum, be soak 60min in 15ppt seawater in salinity, treat that its germ-vesicle 100% breaks completely, obtain ovum.The fresh seawater that ovum utilizes salinity to be 15ppt cleans 3 times repeatedly, and ovum is circle or ellipse.
B, acquisition monomer sperm: with the core population in a step for material, filter out the male several 12 in 2-3 age; Afterwards its sperm quality is detected, under filtering out microscope >=individuality of 30% sperm motility, in salinity 15ppt seawater, activate 10min; Microscopy again afterwards, using 4 of >=90% sperm motility individualities as male parent, obtains monomer sperm.
C, process fertilized egg: added by 4 monomer sperms in 4 monomer ovum liquid respectively, have 3-8 sperm to be advisable with each ovum periphery, ovum density is≤5 ten thousand/mL in good time.During this period, temperature is 31.8 DEG C, and salinity is 15ppt.Wherein, from sperm adds, to finding that the time period that 10% first polar body is released is set to A (8min), the time period of releasing from first polar body to discovery 10% second polar body is set to B (10min), and the time period from second polar body to discovery 10% spilting of an egg individuality is set to C (7min).Select at (A+1/3B) time point, namely after fertilization 11.3min utilizes the 6-dimethylaminopurine (6DMAP) of 450 μm of ol/L to process fertilized egg, suppresses the discharge of fertilized egg second polar body; The process duration is (B+C), is positioned in normal seawater and hatches, namely can obtain 100% Hong Kong oyster triploid D shape larva after namely processing 17min.
D, detection larva ploidy: carrying out Ploidy detection for obtaining first D shape larva employing flow cytometer of incubating in step c, finding that all larvas are all in triploid state, producing without dliploid and aneuploid.After this, all-triploid filial generation is raised according to the conventional breeding method of these species.
Claims (3)
1. produce a time point quantitative analysis method for Hong Kong oyster all-triploid, it is characterized in that, comprise the following steps:
A, obtain monomer ovum: in Hong Kong oyster mating season, using same geographical population as core population, with this core population for material, choose ripe 3-5 Hong Kong in age oyster female individuals; Select with ovum quality and quantity dual indexes, pick out and grow neatly, ten thousand, ovum quantity >=5000, egg membrane is complete, and yolk accumulation is rendered as the individuality of buff as female parent; Collecting monomer ovum, is soak 30-60min in 15-20ppt seawater in salinity, after its germinal vesicle breakdown is complete, then is 15-20ppt seawater cleaning ovum by fresh salinity, obtains monomer ovum liquid;
B, acquisition monomer sperm: with the core population in a step for material, filter out the male in 2-3 age; Afterwards its sperm quality is detected, under filtering out microscope >=individuality of 30% sperm motility, in salinity 15-20ppt seawater, activate 10-15min; Microscopy again afterwards, using the individuality of >=90% sperm motility as male parent, collects monomer sperm;
C, in good time process fertilized egg: monomer sperm is added in monomer ovum liquid, control each ovum periphery and have 3-8 sperm, ovum density domination is at≤5 ten thousand/mL, during this period, temperature controls at 25-32 DEG C, salinity controls at 15-24ppt, wherein, from sperm adds, to finding that the time period that 10% first polar body is released is set to A, the time period of releasing from first polar body to discovery 10% second polar body is set to B, time period from second polar body to discovery 10% spilting of an egg individuality is set to C, select to adopt conventional method process fertilized egg at (A+1/3B) time point, suppress the discharge of fertilized egg second polar body, the process duration is (B+C), be positioned in normal seawater after complete and hatched, namely Hong Kong oyster triploid larva can be obtained.
2. the time point quantitative analysis method of production Hong Kong according to claim 1 oyster all-triploid, is characterized in that, described conventional method process fertilized egg refers to and utilizes cytochalasin B or 6-dimethylaminopurine process fertilized egg.
3. the time point quantitative analysis method of production Hong Kong according to claim 1 oyster all-triploid, is characterized in that, described is 15-20ppt seawater cleaning ovum by fresh salinity, and wash number is 2-3 time.
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| CN108040938B (en) * | 2017-10-09 | 2020-04-03 | 中国科学院南海海洋研究所 | Method for improving production performance of crassostrea hongkongensis triploid through parent improvement |
| CN110055213A (en) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | A kind of separation method of dwarf clam egg membrane |
| CN114208735B (en) * | 2021-12-22 | 2023-02-28 | 中国科学院南海海洋研究所 | Method for cultivating rapid-growth new strain of hong Kong oyster triploid by backcross breeding technology |
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| US7013836B1 (en) * | 2002-06-27 | 2006-03-21 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Enhancing maturation of oocytes in bivalves |
| CN101077063B (en) * | 2006-05-26 | 2012-04-25 | 中国科学院海洋研究所 | Method for breeding triploid monomer oyster in scale |
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