CN109220902A - A kind of producing method for seed of hydrostatic pressing induction rainbow trout triploid - Google Patents
A kind of producing method for seed of hydrostatic pressing induction rainbow trout triploid Download PDFInfo
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- CN109220902A CN109220902A CN201811120871.1A CN201811120871A CN109220902A CN 109220902 A CN109220902 A CN 109220902A CN 201811120871 A CN201811120871 A CN 201811120871A CN 109220902 A CN109220902 A CN 109220902A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
- A01K61/17—Hatching, e.g. incubators
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a kind of producing method for seed of hydrostatic pressing induction rainbow trout triploid, opportune moment after essence, ovum combine, the hydrostatic pressing that regular hour section carries out some strength to fertilized eggs is chosen to handle, then it then returns to and carries out conventional incubation under normal environment, make the fertilized eggs that should be bred as diploid, 100% triploid zygote is generated after hydrostatic pressing is handled, then triploid seed is bred as by triploid zygote.The beneficial effects of the invention are as follows the rainbow trout technical method of triploid seed production of foundation can efficiently, mass induction generate Triploid Rainbow Trout fry, the inductivity of rainbow trout triploid is up to 100%, and there was no significant difference with diploid rainbow trout for survival rate.Method of the invention is easy to operate, practical, easy, safe and reliable, and the large-scale production for rainbow trout triploid provides technical support.
Description
Technical field
The invention belongs to Fish Polyploid Breeding technical fields, are related to a kind of production of hybrid seeds side of hydrostatic pressing induction rainbow trout triploid
Method, the technical method for inducing fertilized ovum chromosome group to double mass production rainbow trout triploid fry by establishing hydrostatic platen press, really
Fixed optimal inductive condition, achievees the purpose that industrialization production infertility triploid, to make up the deficiencies in the prior art.
Background technique
Rainbow trout (Onchorynchus mykiss) be subordinate to salmon shape mesh (Salmoniformes), salmonidae (Salmonidae),
Dog salmon category (Onchorynchus) is gained the name because its sexal maturity individual side has a wide and bright-coloured rainbow band, English
Entitled rainbow trout, originates in north America region, is typical cold water fishes, is worldwide Important Economic cultured fishes
One of and aquatic products genetic breeding field important research object.China starts to introduce and cultivates rainbow trout nineteen fifty-nine, due to long-term
Inbreeding and the breeding for lacking system, lead to cultivate that sex premature occurs in individual, individual smalltype, disease take place frequently etc. asks
Topic, is especially influenced by the big specification salmon of import in recent years, and small dimension salmon trout is very low in the market price of China, supports trout
Person can only import infertility, big individual complete female Triploid Rainbow Trout eyed eggs cultivated to meet market demands because we
Do not have the ability of large-scale production female Triploid Rainbow Trout entirely at present.As it can be seen that the large-scale production of complete female Triploid Rainbow Trout ovum is
Bottleneck as rainbow trout aquaculture.
Triploid Rainbow Trout female individuals are completely sterile, and fish body growth performance and meat reduce caused by avoiding because of gonad development
Risk, culture efficiency is higher；Meanwhile sterile Triploid Rainbow Trout escapes into not will form population in natural water area, Ke Yiwei
The phenomena of mortality held the stabilization of fishery biologic environment, while the mature individual of sexual gland being avoided often to have, have market and the dual effect of ecology
Benefit.Currently, common fish Polyploid Induction Methods include direct method, i.e., directly lured using bioanalysis, physical method and chemical method
Triploid fish are led, wherein the Temperature shock in physical method and hydrostatic platen press are the most commonly used；Indirect method refer to tetraploid with
Diploid hybrid produces triploid induction method, and indirect method requires to obtain a certain number of tetraploid rainbow trouts group, has correlation to grind
Study carefully and show that the survival rate of Tetraploid offspring is not high, therefore it is raw sustainedly and stably to carry out rainbow trout triploid on a large scale
It produces.Based on a large amount of reports, it is Temperature shock, Temperature Treatment ratio that China, which uses at most, in direct method induction rainbow trout triploid
It is easier, it is not necessarily to special equipment, but the highest Triploid Rainbow Trout inductivity of studies in China is 80%, can not successfully realize 100%
Inductivity, and heat shock processing and it is unstable, it is larger to the damage of fertilized eggs so that survival rate is not high, with heat shock
It compares, hydrostatic platen press is relatively small to the stimulation of fertilized eggs, and the processing time is shorter, and also more stable, effect is better than heat shock.So
And China does not still form more mature rainbow trout multiploid induction technical system at present.
Therefore, based on above-mentioned problem, the present invention provides a kind of Triploid Rainbow Trout seeding technique method, passes through hydrostatic platen press
Triploid Rainbow Trout inductivity, survival rate are improved, and establishes the high-quality Seedling production body of the mature and stable polyploid rainbow trout in China oneself
System.
Summary of the invention
The purpose of the present invention is to provide a kind of hydrostatic pressing induction rainbow trout triploid producing method for seed, the invention solves
Technical problem includes:
1 bony fish egg mother cell is fertilized in the second meiotic division, and second polar body is not discharged at this time, fertilized eggs
Possess a set of chromosome from maternal two sets of chromosomes and male parent, amounts to three sets of chromosomes.The core of triploid induction is
Using the discharge of extraneous physical and chemical factor interference fertilized eggs second polar body, and guarantee that the normal spilting of an egg of fertilized eggs, hair eye and rupture of membranes are incubated
Change.If hydrostatic pressing processing is unable to get triploid zygote after second polar body discharge.Therefore, Triploid Rainbow Trout induces
Key technology be determine inhibit rainbow trout fertilized eggs second polar body release optimum time point, i.e., to fertilized eggs apply hydrostatic pressing at
The optimal initial time of reason.
2 are determining optimal initial time and then by single-factor and orthogonal experiment method to hydrostatic pressing size, duration
Etc. parameters groped and optimized, the parameters such as hydrostatic pressing size and duration directly decide induction intensity size.If luring
It leads that intensity is smaller, is unable to reach the purpose for inhibiting second polar body release, and then inductivity can reduce；It is right if inducing intensity excessive
Fertilized eggs will cause certain damage, influence subsequent normal embryo development procedure, and survival rate reduces.Therefore, it is necessary to establish to close
Suitable induction intensity (hydrostatic pressing size, duration) guarantees while inhibiting second polar body discharge, not to fertilized eggs
It is further development damage.
The technical scheme adopted by the invention is that the opportune moment after essence, ovum combine, chooses regular hour section pair
Fertilized eggs carry out the hydrostatic pressing processing of some strength, then then return to and carry out conventional incubation under normal environment, make to educate
At the fertilized eggs of diploid, 100% triploid zygote is generated after hydrostatic pressing is handled, then be bred as three by triploid zygote
Times body seed.
Three key technique parameters of the invention:
Parameter (1), the initial time of hydrostatic pressing processing: under 10.5 DEG C of water temperatures, 20~40 minutes after artificial insemination start
Hydrostatic pressing processing is carried out to fertilized eggs.
Parameter (2), hydrostatic pressing size: 65~68.9kg/cm2。
Parameter (3), hydrostatic pressing processing duration: 5 minutes.
Rainbow trout technical method of triploid seed production, comprising the following steps:
Step 1, ovum artificial collection, semen collection are carried out respectively to female, male rainbow trout, by ova collection to the modeling for filling mono-salt isotonic solution
In feed basin, semen collection is into dry plastic tub, and use is female, male parent population quantitative proportion is 2:1；
Step 2, sperm is poured into the plastic tub for filling ovum by artificial insemination, is gently mixed 15~20 seconds with feather
It is uniformly mixed smart ovum, and starts timing, the starting point as fertilization；
Step 3,3~5 minutes abundant after fertilizations are stood, a little clear water is added repeatedly to wash ovum, ovum is washed and is eliminated as much as in the process
The sundries such as excessive sperm, unfertilized dead ovum and clot；
Step 4,10.5 DEG C are implemented the water swelling of cold flow water 20~40 minutes to fertilized eggs；
Step 5, implement gradient boost method, i.e., fertilized eggs are poured into the homemade plastics keg with permeable mesh, mentioned
Previous minute is put into the compression chamber for filling clear water, every pressurization 10kg/cm2, suspend 10 seconds, to slow down increasing sharply pair for pressure
It is damaged caused by fertilized eggs；
Step 6, implement 65~68.9kg/cm2Pressure size, the duration 5 minutes, prevent the discharge of second polar body,
Induction generates triploid zygote；
Step 7, implement gradient decompression, i.e., every decompression 10kg/cm2, suspend 10 seconds, with slow down pressure reduce rapidly to by
It is damaged caused by smart ovum；
Step 8, implement 10.5 DEG C of cold flow water hatchings to hatching accumulated temperature to subsist up to 220, black eyespot occurs in embryo, in this phase
Between shading treatment is carried out in hatching house, guarantee fertilized eggs subdued light conditions required for incubation period, and reduce the vibration to ovum,
Sterilization processing is carried out to fertilized eggs using methyl blue weekly；
Step 9, implement 10 DEG C of cold flow water to subsist prelarva incubation of membrane to hatching accumulated temperature up to 600；
Step 10, first floating prelarva is implemented under flowing water environment to open the tame and docile feeding of food 30 days, is bred as triploid postlarva.
Wherein, three key parameter determination process of hydrostatic pressing induction rainbow trout triploid are as follows:
Firstly, selection is in 68.9kg/cm2Pressure size, continue 5 minutes induction intensity under, carry out initial time
It determines, initial time selection is in 10~30 minutes ranges, every 2.5 minutes experiment gradients, statistics hair eye rate, Floatation Rate, three
Times body rate.The result shows that each group hair eye rate, Floatation Rate are in rising trend with the extension of initial time, reach in 30 minutes groups
Highest.Initial time each group triploid rate in the range of 20~30 minutes is approximately 100%.Comprehensive hair eye rate, Floatation Rate, three
Times three data of body rate, optimal triploid induction initial time is 30 minutes of after fertilization, under the experiment condition, sends out eye rate
It is 87%, triploid rate 100%.
Secondly, selection is in 65kg/cm2Pressure size, continue 5 minutes induction intensity under, carry out initial time really
Fixed, initial time selected within the scope of 10~40 minutes, every 2.5 minutes experiment gradients, statistics hair eye rate, Floatation Rate, three
Times body rate.The result shows that each group hair eye rate and Floatation Rate reach highest within the scope of initial time 30~35 minutes, remove
Begin 27.5 minutes time processing group, and approximate 100% triploid induction rate can be obtained in initial time 20~40 minutes.It is comprehensive
Eye rate, three Floatation Rate, triploid rate data are sent out, optimal triploid induction initial time is 30~35 minutes of after fertilization.
Finally, rainbow trout fertilized eggs are applied (60,65,70kg/ by the different time (20,30,40 minutes) after selective fertilization
cm2) pressure size, the duration is set as (3,5,7 minutes), statistics hair eye rate, Floatation Rate and triploid rate.It was found that each group is all
Can obtain 100% triploid induction rate, but survival rate influenced by initial time and pressure size it is significant.Initial time
30,40 minutes survival rate no significant differences, and be better than 20 minutes；65,70kg/cm2Hair eye rate no significant difference, still
Filial generation survival rate is significantly lower than 60kg/cm2。
To sum up, the optimum inductive condition of rainbow trout triploid fry is that fertilized eggs are placed on 65 at after fertilization 30~35 minutes
~68.9kg/cm2It is persistently handled in the environment of pressure 5 minutes.
Two, the identification of triploid fry
The identification method of Triploid Rainbow Trout, comprising: (1) triploid is identified by DNA content；(2) pass through measurement red blood cell
Size identifies triploid.
1 identifies triploid by DNA content
The Ploidy Identification that Triploid Rainbow Trout is carried out using flow cytometer (Beckman Coulter FC500M), using just
Normal diploid rainbow trout compares, and determines ploidy, Triploid Rainbow Trout red blood cell by relative dna content in analysis erythrocyte nuclei
DNA content be 1.5 times or so of diploid；Specific implementation step includes:
(1) preparation of single cell suspension: the caudal peduncle of larval rainbow is cut, the PBS for filling 1mL0.01M is rapidly inserted into
In the molecule pipe of buffer solution (pH7.2-7.4), fish body is gently squeezed, flows out blood slowly, amount for taking blood is in 10 μ L or so, so
After shake up, be centrifuged 10s at 3,000 rpm, wash haemocyte, outwell PBS, add the PBS of 1mL, mix.
(2) fixation of single cell suspension: single cell suspension is drawn using disposable plastic tube, it is pre- to be slowly dropped to 3mL
In cold dehydrated alcohol, 4 DEG C save (at least fixed 3h) overnight.
(3) it dyes: the cell suspension 2000rpm being collected into is centrifuged 10min, abandon supernatant, PBS is washed 2 times, microscope
It is lower that cell concentration is adjusted to 1 × 106A/mL, is added 20 μ L, and the RNA enzyme of 50 μ g/mL adds 20-50 μ L, 1g/L after 30min
PI dye liquor, be placed in 4 DEG C of refrigerators and dye 30min, use 300 mesh silk cover filterings.
(4) machine testing on, cell number > 10 of each pattern detection4It is a, resulting data and result are measured by computer
It is handled.
The measurement of 2 erythrocyte sizes
Caudal peduncle arterial blood is extracted, haemocyte smear is made, measures erythrocyte size under the microscope, diploid rainbow trout is red
About 16 μm of cell major diameter average value, about 20 μm of Triploid Rainbow Trout red blood cell major diameter average value.
Beneficial effects of the present invention: the present invention implements hydrostatic pressing processing to rainbow trout fertilized eggs after artificial insemination, prevents
The release of fertilized eggs second polar body successfully obtains 100% Triploid Rainbow Trout seed.The present invention is established using hydrostatic platen press batch
The method of metaplasia production rainbow trout triploid, it is determined that the ginseng such as suitable initial time, pressure size and duration of triploid induction
Number；And the method for establishing the identification of triploid fry.This method is easy to operate, short processing time, lower to the damage of fertilized eggs,
High survival rate.
Detailed description of the invention
Fig. 1 is each group hair eye rate, Floatation Rate；
Fig. 2 is diploid rainbow trout flow cytomery result；
Fig. 3 is Triploid Rainbow Trout flow cytomery result；
Fig. 4 is each experimental group triploid rate；
Fig. 5 is Triploid Rainbow Trout red blood cell (40 × 10)；
Fig. 6 is diploid rainbow trout red blood cell (40 × 10)；
Fig. 7 is hair eye rate；
Fig. 8 is each experimental group triploid rate；
Fig. 9 is each group hair eye rate, Floatation Rate.
Figure 10 is each experimental group triploid rate.
Specific embodiment
The present invention is described in detail With reference to embodiment.
Embodiment 1: rainbow trout technical method of triploid seed production
The seeding technique method the following steps are included:
1 materials and methods
Experiments of single factor is taken, under the conditions of 10.5 DEG C of water temperature, within the scope of 10~30 minutes of after fertilization, if
It sets 9 gradients: initial time 10,12.5,15,17.5,20,22.5,25,27.5,30 minutes, being respectively designated as 31
39 groups, choose 68.9kg/cm2Hydrostatic pressure size, the induction intensity for continuing 5 minutes carry out the induction of rainbow trout triploid, later
Normally hatched, floated, seed rearing, counting the indexs such as each group hair eye rate, Floatation Rate, triploid rate respectively.
2 experimental subjects
In on November 30th, 2017, experiment place was that Weifang City of Shandong Province Linqu salmon trout Fisheries Development is limited for this experiment
Company, using 3,4 age diploid rainbow trout parent populations as experimental subjects, choose individual it is larger, healthy without wound, gonad development is good and vigor
Strong male and female parent population carries out artificial insemination, female-male proportion 2:1.Using wet process insemination method, by ova collection to filling mono-salt
In the plastic tub of isotonic solution, sperm is adopted in dry plastic tub, and sperm is poured into the plastic tub for filling ovum, uses feather
Being gently mixed is uniformly mixed smart ovum, and starts timing, as the starting point of fertilization, stands 3-5min, adds a little clear
Water repeatedly washes ovum, washes ovum and removes the sundries such as excessive sperm, unfertilized dead ovum and clot as far as possible in the process.
3 triploid induction steps
The induction of rainbow trout triploid is carried out using hydrostatic platen press, i.e., some strength is carried out to fertilized eggs using hydrostatic pressing machine
Hydrostatic pressing processing.Until set initial time is poured into fertilized eggs in the homemade plastics keg permeable with mesh, every group
It with ovum amount about 2000, is put into the compression chamber for filling clear water within one minute in advance, progress gradient boost, i.e., every pressurization 10Mpa, temporarily
Stop 10 seconds, to reduce damage of the pressure to fertilized eggs, wait rise to both constant-pressure 68.9kg/cm2Afterwards, start timing, continue 5 minutes
Afterwards, then gradually gradient is depressurized to normal pressure, is then placed in homemade hatching barrel and carries out conventional Lotic hatching, carries out in hatching house
Shading treatment guarantees fertilized eggs subdued light conditions required for incubation period, and reduces vibration, weekly using methyl blue to fertilization
Ovum carries out sterilization processing, periodically chooses except the dead ovum to whiten；Hatching accumulated temperature reaches 220 and subsists, after there is the eyespot of black in embryo, system
Meter hair eye rate (Fig. 1)；When hatching accumulated temperature reaches 600 and subsists, prelarva incubation of membrane is counted Floatation Rate (Fig. 1)；Until prelarva grows to
When 3~4 ㎝, triploid rate is counted.
The identification of 4 Triploid Rainbow Trout fries
Main technical content includes: 1, identifies rainbow trout triploid fry by DNA content；2, pass through measurement erythrocyte size
Identify triploid fry.
1 identifies triploid by DNA content
The Ploidy Identification that Triploid Rainbow Trout is carried out using flow cytometer (Beckman Coulter FC500M), using just
Normal diploid rainbow trout compares, and determines ploidy, Triploid Rainbow Trout red blood cell by relative dna content in analysis erythrocyte nuclei
DNA content be 1.5 times or so of diploid；Specific implementation step includes:
(1) preparation of single cell suspension: the caudal peduncle of larval rainbow is cut, the PBS for filling 1mL0.01M is rapidly inserted into
In the molecule pipe of buffer solution (pH7.2-7.4), fish body is gently squeezed, flows out blood slowly, amount for taking blood is in 10 μ L or so, so
After shake up, be centrifuged 10s at 3,000 rpm, wash haemocyte, outwell PBS, add the PBS of 1mL, mix.
(2) fixation of single cell suspension: single cell suspension is drawn using disposable plastic tube, it is pre- to be slowly dropped to 3mL
In cold dehydrated alcohol, 4 DEG C save (at least fixed 3h) overnight.
(3) it dyes: the cell suspension 2000rpm being collected into is centrifuged 10min, abandon supernatant, PBS is washed 2 times, microscope
It is lower that cell concentration is adjusted to 1 × 106A/mL, is added 20 μ L, and the RNA enzyme of 50 μ g/mL adds 20-50 μ L, 1g/L after 30min
PI dye liquor, be placed in 4 DEG C of refrigerators and dye 30min, use 300 mesh silk cover filterings.
(4) machine testing on, cell number > 10 of each pattern detection4It is a, resulting data and result are measured by computer
It is handled.
The results show that common diploid rainbow trout fluorescent value is 310 ± 5 (Fig. 2), Triploid Rainbow Trout is 437 ± 13 (Fig. 3),
The two ratio is 1.4, close with theoretical value 1.5.Every group randomly selects at least 24 tail fries and carries out Ploidy Identification, counts each group three
Times body rate.As a result, it has been found that the triploid rate of approximation 100% can be obtained within the scope of 20~30 minutes of initial time.Rainbow
The technical requirements of trout triploid mass production guarantee that embryo has certain survival again while inductivity reaches 100%
Rate, from each group send out eye rate and Floatation Rate data it is found that with initial time extension, each group sends out eye rate, Floatation Rate in rising
Trend reaches highest in 30 minutes groups.Therefore the hair eye rate in Comprehensive Experiment result, three Floatation Rate, triploid rate data, can
Using the best initial time for determining Triploid Rainbow Trout induction as 30 minutes of after fertilization, induce in the time available
100% triploid rate, and the hair eye rate of the group and Floatation Rate are relative to control group there are no significant difference.Fig. 4 is each reality
Test a group triploid rate.
The measurement of 2 erythrocyte sizes
Caudal peduncle arterial blood is extracted, haemocyte smear is made, measures erythrocyte size under the microscope, diploid rainbow trout is red
About 16 μm of cell major diameter average value (Fig. 5), about 20 μm of average value of Triploid Rainbow Trout red blood cell major diameter (Fig. 6), Triploid Rainbow Trout is red
Cell long axis is 1.25 times of diploid, is greater than diploid (P < 0.01) extremely significantly, and Triploid Rainbow Trout red cell body is actively shown
It lands and is greater than diploid (P < 0.01), be 1.84 times of diploid, red blood cell area is extremely significant to be greater than diploid (P < 0.01), is
1.49 times of diploid.Triploid and diploid can be accurately and effectively identified by the measurement to rainbow trout erythrocyte size,.
The shortcomings that flow cytometry ploidy is higher to the quality requirement of sample, and sample process process is cumbersome, is needed specific
Instrument, higher cost.The identification method of this method comparison type cell instrument is more convenient, quick, economical.
Embodiment 2: rainbow trout technical method of triploid seed production
The seeding technique method the following steps are included:
1 materials and methods
Experiments of single factor is taken, under the conditions of 10.5 DEG C of water temperature, within the scope of 10~40 minutes of after fertilization, if
It sets 13 gradients: initial time 10,12.5,15,17.5,20,22.5,25,27.5,30,32.5,35,37.5,40 minutes, dividing
It is not named as 313 13 groups, chooses 65kg/cm2Hydrostatic pressure size, the induction intensity for continuing 5 minutes carry out rainbow trout
The induction of triploid is normally hatched later, is floated, seed rearing, is counted each group respectively and is sent out eye rate, Floatation Rate, three times
The indexs such as body rate.
2 experimental subjects
In on December 20th, 2017, experiment place was that Weifang City of Shandong Province Linqu salmon trout Fisheries Development is limited for this experiment
Company, using 3,4 age diploid rainbow trout parent populations as experimental subjects, choose individual it is larger, healthy without wound, gonad development is good and vigor
Strong male and female parent population carries out artificial insemination, female-male proportion 2:1.Using wet process insemination method, by ova collection to filling mono-salt
In the plastic tub of isotonic solution, sperm is adopted in dry plastic tub, and sperm is poured into the plastic tub for filling ovum, uses feather
Being gently mixed is uniformly mixed smart ovum, and starts timing, as the starting point of fertilization, stands 3-5min, adds a little clear
Water repeatedly washes ovum, washes ovum and removes the sundries such as excessive sperm, unfertilized dead ovum and clot as far as possible in the process.
3 triploid induction steps
The induction of rainbow trout triploid is carried out using hydrostatic platen press, i.e., some strength is carried out to fertilized eggs using hydrostatic pressing machine
Hydrostatic pressing processing.Until set initial time is poured into fertilized eggs in the homemade plastics keg permeable with mesh, every group
It with ovum amount about 2000, is put into the compression chamber for filling clear water within one minute in advance, progress gradient boost, i.e., every pressurization 10Mpa, temporarily
Stop 10 seconds, to reduce damage of the pressure to fertilized eggs, wait rise to both constant-pressure 65kg/cm2Afterwards, start timing, continue 5 minutes
Afterwards, then gradually gradient is depressurized to normal pressure, is then placed in homemade hatching barrel and carries out conventional Lotic hatching, carries out in hatching house
Shading treatment guarantees fertilized eggs subdued light conditions required for incubation period, and reduces vibration, weekly using methyl blue to fertilization
Ovum carries out sterilization processing, periodically chooses except the dead ovum to whiten；Hatching accumulated temperature reaches 220 and subsists, after there is the eyespot of black in embryo, system
Meter hair eye rate (Fig. 7)；When hatching accumulated temperature reaches 600 and subsists, prelarva incubation of membrane is counted Floatation Rate (Fig. 7)；Until prelarva grows to
When 3~4 ㎝, triploid rate is counted.
The identification of 4 Triploid Rainbow Trout fries
Main technical content includes: 1, identifies rainbow trout triploid fry by DNA content；2, pass through measurement erythrocyte size
Identify triploid fry.
1 identifies triploid by DNA content
The Ploidy Identification that Triploid Rainbow Trout is carried out using flow cytometer (Beckman Coulter FC500M), using just
Normal diploid rainbow trout compares, and determines ploidy, Triploid Rainbow Trout red blood cell by relative dna content in analysis erythrocyte nuclei
DNA content be 1.5 times or so of diploid；Specific implementation step includes:
(1) preparation of single cell suspension: the caudal peduncle of larval rainbow is cut, the PBS for filling 1mL0.01M is rapidly inserted into
In the molecule pipe of buffer solution (pH7.2-7.4), fish body is gently squeezed, flows out blood slowly, amount for taking blood is in 10 μ L or so, so
After shake up, be centrifuged 10s at 3,000 rpm, wash haemocyte, outwell PBS, add the PBS of 1mL, mix.
(2) fixation of single cell suspension: single cell suspension is drawn using disposable plastic tube, it is pre- to be slowly dropped to 3mL
In cold dehydrated alcohol, 4 DEG C save (at least fixed 3h) overnight.
(3) it dyes: the cell suspension 2000rpm being collected into is centrifuged 10min, abandon supernatant, PBS is washed 2 times, microscope
It is lower that cell concentration is adjusted to 1 × 106A/mL, is added 20 μ L, and the RNA enzyme of 50 μ g/mL adds 20-50 μ L, 1g/L after 30min
PI dye liquor, be placed in 4 DEG C of refrigerators and dye 30min, use 300 mesh silk cover filterings.
(4) machine testing on, cell number > 10 of each pattern detection4It is a, resulting data and result are measured by computer
It is handled.
The results show that common diploid rainbow trout fluorescent value is 310 ± 5, Triploid Rainbow Trout is 437 ± 13, and the two ratio is
1.4, it is close with theoretical value 1.5.Every group randomly selects at least 24 tail fries and carries out Ploidy Identification, counts each group triploid rate.Knot
Fruit shows that within the scope of initial time 30~35 minutes, each group hair eye rate and Floatation Rate reach highest, removes initial time 27.5
Minute processing group can obtain approximate 100% triploid induction rate in initial time 20~40 minutes.Comprehensive hair eye rate, on
Floating three rate, triploid rate data, optimal triploid induction initial time are 30~35 minutes of after fertilization.
The measurement of 2 erythrocyte sizes
Caudal peduncle arterial blood is extracted, haemocyte smear is made, measures erythrocyte size under the microscope, diploid rainbow trout is red
About 16 μm of cell major diameter average value, about 20 μm of Triploid Rainbow Trout red blood cell major diameter average value, Triploid Rainbow Trout red blood cell long axis is
1.25 times of diploid are greater than diploid (P < 0.01) extremely significantly, and Triploid Rainbow Trout red cell body is actively greater than two significantly
Times body (P < 0.01) is 1.84 times of diploid, and it is diploid that red blood cell area is extremely significant, which to be greater than diploid (P < 0.01),
1.49 again.Triploid and diploid can be accurately and effectively identified by the measurement to rainbow trout erythrocyte size,.Fluidic cell
Instrument identifies that the shortcomings that ploidy is higher to the quality requirement of sample, and sample process process is cumbersome, needs specific instrument, at
This is higher.The identification method of this method comparison type cell instrument is more convenient, quick, economical.
Embodiment 3: rainbow trout technical method of triploid seed production
The seeding technique method the following steps are included:
1 materials and methods
Take orthogonal experiment method, to influence the initial time of experimental result, hydrostatic pressing size, the duration three it is main because
Element is tested, and each factor is arranged three levels, respectively initial time (20,30,40 minutes), pressure size (60,
65、70kg/cm2), the duration (3,5,7 minutes) (table 1).Normally hatched under the conditions of 10.5 DEG C of water temperature, floated,
Seed rearing counts the indexs such as each group hair eye rate, Floatation Rate, triploid rate respectively.
1 Triploid Rainbow Trout of table induces orthogonal experiment scheme
2 experimental subjects
In on January 10th, 2017, experiment place was that Weifang City of Shandong Province Linqu salmon trout Fisheries Development is limited for this experiment
Company, using 3,4 age diploid rainbow trout parent populations as experimental subjects, choose individual it is larger, healthy without wound, gonad development is good and vigor
Strong male and female parent population carries out artificial insemination, female-male proportion 2:1.Using wet process insemination method, by ova collection to filling mono-salt
In the plastic tub of isotonic solution, sperm is adopted in dry plastic tub, and sperm is poured into the plastic tub for filling ovum, uses feather
Being gently mixed is uniformly mixed smart ovum, and starts timing, as the starting point of fertilization, stands 3-5min, adds a little clear
Water repeatedly washes ovum, washes ovum and removes the sundries such as excessive sperm, unfertilized dead ovum and clot as far as possible in the process.
3 triploid induction steps
The induction of rainbow trout triploid is carried out using hydrostatic platen press, i.e., some strength is carried out to fertilized eggs using hydrostatic pressing machine
Hydrostatic pressing processing.Until set initial time is poured into fertilized eggs in the homemade plastics keg permeable with mesh, every group
It with ovum amount about 2000, is put into the compression chamber for filling clear water within one minute in advance, progress gradient boost, i.e., every pressurization 10Mpa, temporarily
Stop 10 seconds, to reduce damage of the pressure to fertilized eggs, after rising to both constant-pressures, starts timing, after certain time, then by
Step gradient is depressurized to normal pressure, is then placed in homemade hatching barrel and carries out conventional Lotic hatching, carries out at shading in hatching house
Reason guarantees fertilized eggs subdued light conditions required for incubation period, and reduces vibration, is carried out using methyl blue to fertilized eggs weekly
Sterilization processing is periodically chosen except the dead ovum to whiten；Hatching accumulated temperature reaches 220 and subsists, after there is the eyespot of black in embryo, statistics hair eye
Rate (Fig. 9)；When hatching accumulated temperature reaches 600 and subsists, prelarva incubation of membrane is counted Floatation Rate (Fig. 9)；Until prelarva grows to 3~4 ㎝
When, count triploid rate.
The identification of 4 Triploid Rainbow Trout fries
Main technical content includes: 1, identifies rainbow trout triploid fry by DNA content；2, pass through measurement erythrocyte size
Identify triploid fry.
1 identifies triploid by DNA content
The Ploidy Identification that Triploid Rainbow Trout is carried out using flow cytometer (Beckman Coulter FC500M), using just
Normal diploid rainbow trout compares, and determines ploidy, Triploid Rainbow Trout red blood cell by relative dna content in analysis erythrocyte nuclei
DNA content be 1.5 times or so of diploid；Specific implementation step includes:
(1) preparation of single cell suspension: the caudal peduncle of larval rainbow is cut, the PBS for filling 1mL0.01M is rapidly inserted into
In the molecule pipe of buffer solution (pH7.2-7.4), fish body is gently squeezed, flows out blood slowly, amount for taking blood is in 10 μ L or so, so
After shake up, be centrifuged 10s at 3,000 rpm, wash haemocyte, outwell PBS, add the PBS of 1mL, mix.
(2) fixation of single cell suspension: single cell suspension is drawn using disposable plastic tube, it is pre- to be slowly dropped to 3mL
In cold dehydrated alcohol, 4 DEG C save (at least fixed 3h) overnight.
(3) it dyes: the cell suspension 2000rpm being collected into is centrifuged 10min, abandon supernatant, PBS is washed 2 times, microscope
It is lower that cell concentration is adjusted to 1 × 106A/mL, is added 20 μ L, and the RNA enzyme of 50 μ g/mL adds 20-50 μ L, 1g/L after 30min
PI dye liquor, be placed in 4 DEG C of refrigerators and dye 30min, use 300 mesh silk cover filterings.
(4) machine testing on, cell number > 10 of each pattern detection4It is a, resulting data and result are measured by computer
It is handled.
It can be seen that three kinds of initial time, pressure size, duration empirical factors from each group hair eye rate, Floatation Rate
Very poor is respectively 16.2%, 14.5%, 1.1%, it can be seen that the principal element for influencing experiment is respectively initial time and pressure
Size, influence of the duration to result is little, and the average hair eye rate between more same factor different level can be seen that
30, the hair eye rate no significant difference of 40min, and it is better than 20min.65,70kg/cm2Induced pressure no significant difference, still
Filial generation survival rate is significantly lower than 60kg/cm2, the duration of 3~7min is to survival rate no significant difference.
The results show that common diploid rainbow trout fluorescent value is 310 ± 5, Triploid Rainbow Trout is 437 ± 13, and the two ratio is
1.4, it is close with theoretical value 1.5.Every group randomly selects at least 24 tail fries and carries out Ploidy Identification, counts each group triploid rate.It removes
First group for going unexpected death, each group triploid rate is all 100%, shows 20~40min, 60~70Mpa, 3~7min is to three
The influence of times body rate is simultaneously little, in this time frame in induction can obtain 100% triploid rate.
The measurement of 2 erythrocyte sizes
Caudal peduncle arterial blood is extracted, haemocyte smear is made, measures erythrocyte size under the microscope, diploid rainbow trout is red
About 16 μm of cell major diameter average value, about 20 μm of Triploid Rainbow Trout red blood cell major diameter average value, Triploid Rainbow Trout red blood cell long axis is
1.25 times of diploid are greater than diploid (P < 0.01) extremely significantly, and Triploid Rainbow Trout red cell body is actively greater than two significantly
Times body (P < 0.01) is 1.84 times of diploid, and it is diploid that red blood cell area is extremely significant, which to be greater than diploid (P < 0.01),
1.49 again.Triploid and diploid can be accurately and effectively identified by the measurement to rainbow trout erythrocyte size,.Fluidic cell
Instrument identifies that the shortcomings that ploidy is higher to the quality requirement of sample, and sample process process is cumbersome, needs specific instrument, at
This is higher.The identification method of this method comparison type cell instrument is more convenient, quick, economical.
The above is only not to make limit in any form to the present invention to better embodiment of the invention
System, any simple modification that embodiment of above is made according to the technical essence of the invention, equivalent variations and modification,
Belong in the range of technical solution of the present invention.
Claims (4)
1. a kind of producing method for seed of hydrostatic pressing induction rainbow trout triploid, it is characterised in that: in appropriate after rainbow trout essence, ovum combine
Machine is chosen the hydrostatic pressing that regular hour section carries out some strength to fertilized eggs and is handled, then then returns to normal environment
Lower carry out conventional incubation, makes the fertilized eggs that should be bred as diploid, and 100% triploid fertilization is generated after hydrostatic pressing is handled
Ovum, then triploid seed is bred as by triploid zygote.
2. according to a kind of producing method for seed of the induction rainbow trout triploid of hydrostatic pressing described in claim 1, it is characterised in that: the hydrostatic
It presses the initial time of processing: under 10.5 DEG C of water temperatures, starting within 20~40 minutes after artificial insemination to carry out at hydrostatic pressing fertilized eggs
Reason, hydrostatic pressing size: 65~68.9kg/cm2, hydrostatic pressing handle the duration: 5 minutes.
3. according to a kind of producing method for seed of the induction rainbow trout triploid of hydrostatic pressing described in claim 1, it is characterised in that: described three times
System kind technical method, comprising the following steps:
Step 1, ovum artificial collection, semen collection are carried out respectively to female, male rainbow trout, by ova collection to the plastic tub for filling mono-salt isotonic solution
In, semen collection is into dry plastic tub, and use is female, male parent population quantitative proportion is 2:1；
Step 2, sperm is poured into the plastic tub for filling ovum by artificial insemination, and being gently mixed 15~20 seconds with feather makes essence
Ovum is uniformly mixed, and starts timing, the starting point as fertilization；
Step 3,3~5 minutes abundant after fertilizations are stood, a little clear water is added repeatedly to wash ovum, ovum is washed and is eliminated as much as excess in the process
The sundries such as sperm, unfertilized dead ovum and clot；
Step 4,10.5 DEG C are implemented the water swelling of cold flow water 20~40 minutes to fertilized eggs；
Step 5, implement gradient boost method, i.e., pour into fertilized eggs in the homemade plastics keg with permeable mesh, in advance one
Minute is put into the compression chamber for filling clear water, every pressurization 10kg/cm2, suspend 10 seconds, to slow down the increasing sharply to fertilization of pressure
It is damaged caused by ovum；
Step 6, implement 65~68.9kg/cm2Pressure size, the duration 5 minutes, prevent the discharge of second polar body, induction produces
Raw triploid zygote；
Step 7, implement gradient decompression, i.e., every decompression 10kg/cm2, suspend 10 seconds, to slow down the reducing rapidly to fertilized eggs of pressure
Caused by damage；
Step 8, implement 10.5 DEG C of cold flow water hatchings to hatching accumulated temperature to subsist up to 220, black eyespot occurs in embryo, incubates during this period
Change indoor carry out shading treatment, guarantees fertilized eggs subdued light conditions required for incubation period, and reduce the vibration to ovum, weekly
Sterilization processing is carried out to fertilized eggs using methyl blue；
Step 9, implement 10 DEG C of cold flow water to subsist prelarva incubation of membrane to hatching accumulated temperature up to 600；
Step 10, first floating prelarva is implemented under flowing water environment to open the tame and docile feeding of food 30 days, is bred as triploid postlarva.
4. according to a kind of producing method for seed of the induction rainbow trout triploid of hydrostatic pressing described in claim 3, it is characterised in that: described three times
The identification of body fry:
(1) triploid is identified by DNA content
The Ploidy Identification that Triploid Rainbow Trout is carried out using flow cytometer (Beckman Coulter FC500M), using normal two
Times body rainbow trout compares, and determines ploidy by relative dna content in analysis erythrocyte nuclei, Triploid Rainbow Trout red blood cell
DNA content is 1.5 times or so of diploid；
(2) triploid is identified by measurement erythrocyte size
Caudal peduncle arterial blood is extracted, haemocyte smear is made, measures erythrocyte size, diploid rainbow trout red blood cell under the microscope
About 16 μm of major diameter average value, about 20 μm of Triploid Rainbow Trout red blood cell major diameter average value.
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