CN109874707A - A kind of method of efficient initiative allooctaploid pond crucian carp - Google Patents
A kind of method of efficient initiative allooctaploid pond crucian carp Download PDFInfo
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- CN109874707A CN109874707A CN201910274442.8A CN201910274442A CN109874707A CN 109874707 A CN109874707 A CN 109874707A CN 201910274442 A CN201910274442 A CN 201910274442A CN 109874707 A CN109874707 A CN 109874707A
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Abstract
The present invention relates to a kind of methods of efficiently initiative allooctaploid pond crucian carp.This method includes the artificial insemination stage, and the artificial insemination stage includes: configuration trypsin solution: trypsase powder being added in spermatozoa preservative fluid and mixes acquisition trypsin solution, trypsase powder 0.9-1.1g is added in every 100mL spermatozoa preservative fluid；Liquefacient duration: the mature sperm of white crucian carp is added in above-mentioned trypsin solution and handles 13-17min under the conditions of 22-24 DEG C；Insemination hatching: the mature egg of treated sperm and hybridized prussian carp is subjected to dry method insemination, gained fertilized eggs carry out hatching and obtain allooctaploid pond crucian carp.The present invention handles sperm by the trypsin solution that spermatozoa preservative fluid dissolves, keep sperm solution solidifying, realize that Heterologous Sperm is successfully integrated into hybridized prussian carp ovum, form allooctaploid, and by the suitable trypsinase concentration of setting and processing time, to further obtain a high proportion of allooctaploid pond crucian carp.
Description
Technical field
The invention belongs to genetics-breeding in fish fields, are specifically related to a kind of side of efficiently initiative allooctaploid pond crucian carp
Method.
Background technique
Hybridization and polyploidization can provide original power and material base for spore and merit formation.From reason
By upper theory, due to the increase of genome ploidy, often cell size can increase therewith, and polyploid usually shows closer than diploid
Faster, nutrition organs or individual are bigger for one's own length.As a kind of classical and fruitful cell engineering breeding technique, more times
Sport kind (polyploid breeding) has widely applied in animals and plants breeding, including the product in cultured fishes and shellfish
In kind breeding.In aquiculture animal, successful example has Crassostrea gigas triploidy and natural from Cyprinidae and salmonidae etc.
The homologous or allopolyploid synthesized in polyploid fish.Therefore, optimize or develop new, efficient ploidy operating technology, wound
Excellent germplasm processed provides core breeding material for rearing new variety, is still the important directions of aquiculture animal genetic breeding
One of.
Hybridized prussian carp (Carassius gibelio Bloch) is important one of the large freshwater aquiculture type in China, closely
Gross annual output amount has been approached 3,000,000 tons together for several years pond crucian carps and other crucians.Pond crucian carp can be carried out Natural Gynogenesiss reproduction, by emerging
State's red cyprinoid is provided in the heterologous pond crucian carp group of sperm artificial breeding, it has been found that it is complete that a small number of (about 0.2%) had both maintained pond crucian carp
Set genome incorporates the genomic artificial synthesized allooctaploid pond crucian carp of a set of red cyprinoid again simultaneously, to present pond crucian carp
Integrate the ability of foreign gene group.This unique ability can formulate excellent germplasm for the breeding of novel carassius auratus gibelio kind.For example, different
Educating pond crucian carp " middle section 5 " is from the breeding core for being infiltrated with megalobrama amblycephala sperm minute chromosome segment, character is substantially change
Group, through continuous ten generations gynogenesis breeding；" Changfeng crucian carp " is that D system pond crucian carp ovum has incorporated a set of Singuo Red Carp sperm
Genome development.But after entering pond crucian carp archiblast due to heterologous sperm, does not understand solidifying and be in pyknosis state, so by different
It is only 0.2-0.33% that smart gynogenesis, which spontaneously forms allooctaploid filial generation digit rate, this also causes the screening and identification in breeding different
Source octoploid workload is very big.
Summary of the invention
In order to solve the above technical problem, the present invention provides a kind of methods of efficiently initiative allooctaploid pond crucian carp.
In order to achieve the object of the present invention, the invention adopts the following technical scheme:
A kind of method of efficient initiative allooctaploid pond crucian carp, including artificial insemination stage, the artificial insemination stage packet
Include following operating procedure:
Step 1, it configures trypsin solution: trypsase powder being added in spermatozoa preservative fluid and mixes acquisition tryptose
The trypsase powder 0.9-1.1g is added in every 100mL spermatozoa preservative fluid in enzyme solutions；
Step 2, liquefacient duration: the mature sperm of white crucian carp is added in above-mentioned trypsin solution and in 22-24 DEG C of item
13-17min is handled under part；
Step 3, insemination hatching: carrying out dry method insemination for the mature egg of the sperm and hybridized prussian carp that handle by step 2,
Gained fertilized eggs carry out hatching and obtain allooctaploid pond crucian carp.
Further technical solution: the sperm storage formula of liquid are as follows: NaCl 7.5-8.5g/L, KCl0.3-0.5g/L,
CaCl20.12-0.16g/L、MgSO4·7H2O 0.1-0.3g/L、Na2HPO4·H2O0.04-0.08g/L、KH2PO40.04-
0.08g/L、NaHCO30.33-0.37g/L, glucose 0.9-1.1g/L；The spermatozoa preservative fluid pH=7.0-7.2.
Further technical solution: the hatching pattern is to be placed in fertilized eggs to hatch in ceramic whiteware disk and change one in every 12 hours
Half water, or fertilized eggs are placed in mesh sheet to be put into water tank and are oxygenated hatching.
Further technical solution: this method further includes hybridized prussian carp parent culture and reproductive stage, the hybridized prussian carp
Parent culture and reproductive stage the following steps are included:
Step 1, pond-inputting prepares: in winter, dry pool disinfection first is carried out to the parent culture pool before parent's pond-inputting, kills trash fish,
Then Xin Shui is poured into again to the parent culture pool, while isolation net cover is set to prevent trash fish to be mixed into parent culture pool water inlet
Cultivate the pool；
Step 2, parent culture: carrying out male and female screening before parent's pond-inputting, eliminates milter；By the parent culture pool depth of water after the beginning of spring
It improves to 1.5-2.0 meters；
The feeding volume of feed is calculated according to the 2-4% of parent's weight total amount during parent culture；
Splash during parent culture into the parent culture pool each 3000-4500kg/hm of decomposed organic fertilizer2Or fermentation
1200 milliliter/mu of EM bacterium solution/7-15 days, reduce Dissolved Oxygen in Water to 2-4mg/L；
When parent culture pool water temperature is at 16-22 DEG C, promote parent's oviposition in the form of seine, when seine avoids to parent
Originally it damages, draws in the net the parent captured by maturity identification, the parent of the development of ovary to fourth phase mid-term and later is stood
It hastens parturition, the parent of the development of ovary to the initial stage fourth phase is turned to support to interior to do induced reproduction preparation；
Step 3, it hastens parturition: using+500 international units of 1ug/kg dioxone+1mg/kg pituitary/kg human chorionic gonadotropin's gland
The mixed liquor of hormone+5ug/kg ovulation induction element carries out injection to parent and hastens parturition, and the parent after injection is hastened parturition is put into constant indoor temperature 21
Oviposition is waited in the cylinder of DEG C flowing water.
Further technical solution: this method further includes the Ploidy detection mode of filial generation after hatching, the Ploidy detection side
Formula the following steps are included:
Step 1, it takes blood: reaching the fry of 1.5cm for length and 0.1 μ L of blood is taken using docking, for reaching six-month-old
Fry use cuts fin and takes 0.1 μ L of blood；
Step 2, it detects: adopted new blood being placed in the CyStain DNA1Step solution that 200 μ L are pre-chilled through 4 DEG C and is mixed
It after even, places on ice, and carries out DNA content detection using flow cytometer.
Further technical solution: white crucian carp male parent is hastened parturition by the dosage injection of above-mentioned mixed liquor one third, the white crucian carp
Male parent is 2 more than age healthy and strong individuals, and weight is in 300-500g.
Further technical solution: it during the parent culture, rises sharply area for will appear temperature March, then in gas
Divide cylinder temporarily to support in 18-22 DEG C of circulating water body indoors parent at warm 16-20 DEG C, then carries out subsequent hasten parturition.
The beneficial effects of the present invention are:
(1) present invention is by spermatozoa preservative fluid, while using trypsin treatment sperm, coagulates sperm solution, realizes heterologous essence
Sub (white crucian carp) is successfully integrated into hybridized prussian carp ovum, forms allooctaploid, and the trypsinase concentration suitable by setting
With the processing time, to further obtain a high proportion of allooctaploid pond crucian carp, allooctaploid formation rate of the present invention is up to
16.8%, the survival rate of allooctaploid pond crucian carp is 2.4 ± 0.7%.
(2) spermatozoa preservative fluid of the present invention sterilizing H2O configuration, can both use after the sperm storage formula of liquid is modulated
In preservation sperm, and trypsase can be made to play activity.
(3) about hatching pattern:
The mesh sheet is stainless steel frame, and tow sides are close eye mesh sheet, and material is generally polyethylene.Fish-egg is adhered to
After in mesh sheet, mesh sheet is put into water tank, oxygenation hatching.The advantages of operation is that hatching water body is big, and hatching process does not have to change water.
The ceramic whiteware disk is medical experimental disk, and fish-egg is layered in ceramic whiteware disk, water is added to hatch, changed half every 12 hours
Water.The advantages of hatching method, is: the developmental morphology of fish-egg can be observed at any time, periodically eliminate dead ovum, in order to avoid lose
The hatching of the normal fish-egg of bad water quality impact.
(4) hybridized prussian carp parent culture of the present invention and reproductive stage are to obtain a high proportion of allooctaploid pond crucian carp
Basis, wherein as follows the advantages of each step:
Isolation net cover is set in culture pond water inlet, mainly prevents trash fish to be mixed into and cultivates the pool, to avoid mating period
Trash fish sperm stimulation hybridized prussian carp parent causes to miscarry.
The effect that male and female screening is carried out before parent's pond-inputting is to eliminate milter, in order to avoid milter stimulates raun in spring, cause
Raun miscarriage.The parent culture pool depth of water is improved after the beginning of spring and is to avoid water temperature to rise too fast to 1.5-2.0 meters of effect and leads
Cause parent's miscarriage.
The organic fertilizer or EM bacterium solution for splashing into the parent culture pool decomposed can guarantee there is work abundant in culture pond water
Body bait.It not only ensure that the needs of gonad development of parents about the control for reducing Dissolved Oxygen in Water, but also will not parent done sth. in advance
Miscarriage.
Seine can promote parent to lay eggs, but the mode of " upper layer seine, do not pay attention to baseline " is used when seine, avoid to parent
Originally it causes to stimulate and cause to miscarry.
In In Middle And Lower Reaches of Changjiang River, often occurs unexpected heating in continuous several days in mid or late March, will lead to parent's
Miscarriage.So dividing cylinder temporary parent at 16-20 DEG C of temperature to guarantee the abundant mature of parent and avoid abortion phenomena
It supports in 18-22 DEG C of thermostatical circulating water body indoors.When due to parent culture, abundant living body bait gives enough nutrition, low
Oxygen content amount is not enough to postmaturity again, so that parent temporarily supports the one and a half months time indoors, can still carry out induced reproduction.
The present invention implements to hasten parturition to parent realizes the synchronism of parent's oviposition, so that it is numerous to be conducive to the large-scale seed of development
It grows.Present invention injection hasten parturition after parent to be put into effector phase in the cylinder of 21 DEG C of flowing water of constant indoor temperature be 10 hours or so, be convenient for
The arrangement that subsequent breeding behaviour does.It is of the present invention hasten parturition dosage and hasten parturition temperature the advantages of: avoid the water temperature pairing effect phase
It influences, is easy to hold egg-laying time, the good subsequent breeding operation of reasonable arrangement；The oxytocic drug compatibility, enables to parent's ovum
Maturity it is uniform, for breeding provide good ovum.
(5) Ploidy detection mode amount for taking blood of the present invention is few, cuts fin ray and takes the method for blood easy to operate, and can not kill
Detect obtained octoploid fish.The test sample mixed is put on ice for by the present invention, can store six hours, this is to take
Sample detection provides sufficient time support, while being put on ice for can be improved the inspection success rate of instrument, to mention significantly
High detection efficiency.
Detailed description of the invention
Part A is the aspect graph of female parent hybridized prussian carp A+ system, the white crucian carp of male parent and the new octoploid of pond crucian carp in Fig. 1.
Part B is that female parent hybridized prussian carp A+ system, the white crucian carp of male parent and pond crucian carp allooctaploid peripheral blood DNA content are straight in Fig. 1
Fang Tu.
C portion is female parent hybridized prussian carp A+ system, the white crucian carp of male parent and the division of pond crucian carp allooctaploid mitosis metaphase in Fig. 1
Phasor.
Specific embodiment
More specific detail is made to technical solution of the present invention below with reference to embodiment:
The method that the present invention efficiently formulates allooctaploid pond crucian carp, including hybridized prussian carp parent culture and reproductive stage, people
The Ploidy detection mode of filial generation after work insemination stage and hatching.
About hybridized prussian carp parent culture and reproductive stage:
Embodiment 1
Step 1, pond-inputting prepares: in winter, first carrying out to the parent culture pool before parent (kind: hybridized prussian carp A+ system) pond-inputting
Dry pool disinfection, kills trash fish, then pours into Xin Shui again to the parent culture pool, while being arranged in parent culture pool water inlet and being isolated
Net cover cultivates the pool to prevent trash fish to be mixed into；
Step 2, parent culture: carrying out male and female screening before parent's pond-inputting, eliminates milter；By the parent culture pool depth of water after the beginning of spring
It improves to 1.8 meters；
The feeding volume of feed is calculated according to the 3% of parent's weight total amount during parent culture；
Splash during parent culture into the parent culture pool each 4000kg/hm of decomposed organic fertilizer2, it is molten to reduce water body
Oxygen is solved to 3mg/L；
When parent culture pool water temperature is at 20 DEG C, promote parent's oviposition in the form of seine, when seine avoids to parent
It damages, draws in the net the parent captured by maturity identification, immediately to the parent of the development of ovary to fourth phase mid-term and later
It hastens parturition, the parent of the development of ovary to the initial stage fourth phase is turned to support to interior to do induced reproduction preparation；
Step 3, it hastens parturition: using+500 international units of 1ug/kg dioxone+1mg/kg pituitary/kg human chorionic gonadotropin's gland
The mixed liquor of hormone+5ug/kg ovulation induction element carries out injection to parent and hastens parturition, and the parent after injection is hastened parturition is put into constant indoor temperature 21
Oviposition is waited in the cylinder of DEG C flowing water.White crucian carp male parent (being selected from In Nanning Area) is urged by the dosage injection of above-mentioned mixed liquor one third
It produces, the white crucian carp male parent is 2 more than age healthy and strong individuals, and weight is in 400g or so.
Embodiment 2
Step 1, pond-inputting prepares: in winter, first carrying out to the parent culture pool before parent (kind: hybridized prussian carp A+ system) pond-inputting
Dry pool disinfection, kills trash fish, then pours into Xin Shui again to the parent culture pool, while being arranged in parent culture pool water inlet and being isolated
Net cover cultivates the pool to prevent trash fish to be mixed into；
Step 2, parent culture: carrying out male and female screening before parent's pond-inputting, eliminates milter；By the parent culture pool depth of water after the beginning of spring
It improves to 1.5 meters；
The feeding volume of feed is calculated according to the 2% of parent's weight total amount during parent culture；
Splash during parent culture into the parent culture pool each 3000kg/hm of decomposed organic fertilizer2, it is molten to reduce water body
Oxygen is solved to 4mg/L；
When parent culture pool water temperature is at 16 DEG C, promote parent's oviposition in the form of seine, when seine avoids to parent
It damages, draws in the net the parent captured by maturity identification, immediately to the parent of the development of ovary to fourth phase mid-term and later
It hastens parturition, the parent of the development of ovary to the initial stage fourth phase is turned to support to interior to do induced reproduction preparation；
Step 3, it hastens parturition: using+500 international units of 1ug/kg dioxone+1mg/kg pituitary/kg human chorionic gonadotropin's gland
The mixed liquor of hormone+5ug/kg ovulation induction element carries out injection to parent and hastens parturition, and the parent after injection is hastened parturition is put into constant indoor temperature 21
Oviposition is waited in the cylinder of DEG C flowing water.White crucian carp male parent is hastened parturition by the dosage injection of above-mentioned mixed liquor one third, the white crucian carp male parent
For 2 more than age healthy and strong individuals, weight is in 300g or so.
Embodiment 3
Step 1, pond-inputting prepares: in winter, first carrying out to the parent culture pool before parent (kind: hybridized prussian carp A+ system) pond-inputting
Dry pool disinfection, kills trash fish, then pours into Xin Shui again to the parent culture pool, while being arranged in parent culture pool water inlet and being isolated
Net cover cultivates the pool to prevent trash fish to be mixed into；
Step 2, parent culture: carrying out male and female screening before parent's pond-inputting, eliminates milter；By the parent culture pool depth of water after the beginning of spring
It improves to 2.0 meters；
The feeding volume of feed is calculated according to the 4% of parent's weight total amount during parent culture；
It is splashed during parent culture into the parent culture pool 1200 milliliter/mu/10 day of EM bacterium solution of fermentation, reduces water
Body dissolved oxygen is to 2mg/L；
The present embodiment is implemented in In Middle And Lower Reaches of Changjiang River, and mid or late March temperature occurs and rises sharply, then at 18 DEG C of temperature or so
When divide cylinder temporarily to support indoors in 18-22 DEG C of circulating water body the parent, then carry out subsequent hasten parturition.
Step 3, it hastens parturition: using+500 international units of 1ug/kg dioxone+1mg/kg pituitary/kg human chorionic gonadotropin's gland
The mixed liquor of hormone+5ug/kg ovulation induction element carries out injection to parent and hastens parturition, and the parent after injection is hastened parturition is put into constant indoor temperature 21
Oviposition is waited in the cylinder of DEG C flowing water.White crucian carp male parent is hastened parturition by the dosage injection of above-mentioned mixed liquor one third, the white crucian carp male parent
For 2 more than age healthy and strong individuals, weight is in 500g or so.
Judgment criteria about parent's maturity before hastening parturition is as follows:
Hybridized prussian carp parent (female parent) is general, and abdomen is soft, expands, ovary is clear-cut, and gonopore is micro- red, and gonopore
Very red parent voluntarily miscarries, it should weed out.Sometimes for more accurately identify hybridized prussian carp parent maturity,
It can be used and dig egg apparatus digging ovum inspection.Egg apparatus will be taken slowly to be inserted into gonopore, then to the left or right avertence is a little, rotate it is several under gently
Extraction, that is, can be taken off a small amount of ovum grain, and a small amount of fixed transparent liquid is then added, observes egg nucleus position after impregnating 2-3 minutes.If all
Or the core position bias or polarization of most of ovum grain, then show that parent's maturity is good, is suitble to carry out induced reproduction at once；If white
Nucleus occupies central location, then parent's sexal maturity is poor, also needs further to cultivate mature；If most of ovum grain goes out without the core of white
It is existing, it is generally degeneration ovum, is not suitable for being bred again.
Criterion of the present invention to maternal maturity are as follows: the light abdomen squeezed near maternal gonopore, if the fourth phase squeezed out
Egg mother cell is (shows the maternal development of ovary to fourth phase mid-term and later) of separation, one one, can be hastened parturition；If squeezing
Out be unsegregated pockets of (showing the maternal development of ovary to the initial stage fourth phase), then also need to raise again 7-15 days.
Male parent of the invention, it is more than age that the white crucian carp selected from Nanning is 2,300-500 grams of weight, the healthy and strong pure lines product of individual
Kind.Mature white crucian carp parent generally has apparent " star-pursuing " in pectoral fin, abdomeinal fin and the gill cover, under gonopore is slightly concave, when gently pressing abdomen,
There is the thick liquid semen outflow of milky, shows that milter maturity is preferable.
The artificial insemination stage includes following operating procedure:
Embodiment 4
Step 1, it configures trypsin solution: trypsase powder being added in spermatozoa preservative fluid and mixes acquisition tryptose
The trypsase powder 1.0g is added in every 100mL spermatozoa preservative fluid in enzyme solutions；
The sperm storage formula of liquid are as follows: NaCl 8.0g/L, KCl 0.4g/L, CaCl20.14g/L、MgSO4·7H2O
0.2g/L、Na2HPO4·H2O 0.06g/L、KH2PO40.06g/L、NaHCO30.35g/L, glucose 1.0g/L；The sperm is protected
Liquid storage pH=7.1.
Step 2, liquefacient duration: the mature sperm of white crucian carp is added in above-mentioned trypsin solution and at 20 °C
Handle 15min；
Step 3, insemination hatching: by the sperm handled by step 2 (from white crucian carp in embodiment 1) and hybridized prussian carp
Mature egg (from " hybridized prussian carp A+ system " in embodiment 1) carries out dry method insemination, and it is different that gained fertilized eggs carry out hatching acquisition
Source octoploid pond crucian carp.The hatching pattern is to be placed in fertilized eggs to hatch in ceramic whiteware disk and change half water in every 12 hours.
Embodiment 5
Step 1, it configures trypsin solution: trypsase powder being added in spermatozoa preservative fluid and mixes acquisition tryptose
The trypsase powder 0.9g is added in every 100mL spermatozoa preservative fluid in enzyme solutions；
The sperm storage formula of liquid are as follows: NaCl 7.5g/L, KCl 0.3g/L, CaCl20.12g/L、MgSO4·7H2O
0.1g/L、Na2HPO4·H2O 0.04g/L、KH2PO40.04g/L、NaHCO30.33g/L, glucose 0.9g/L；The sperm is protected
Liquid storage pH=7.0.
Step 2, liquefacient duration: the mature sperm of white crucian carp is added in above-mentioned trypsin solution and under the conditions of 22 DEG C
Handle 13min；
Step 3, insemination hatching: by the sperm handled by step 2 (from white crucian carp in embodiment 2) and hybridized prussian carp
Mature egg (from " hybridized prussian carp A+ system " in embodiment 2) carries out dry method insemination, and it is different that gained fertilized eggs carry out hatching acquisition
Source octoploid pond crucian carp.The hatching pattern is that fertilized eggs are placed in mesh sheet to be put into water tank to be oxygenated hatching.
Embodiment 6
Step 1, it configures trypsin solution: trypsase powder being added in spermatozoa preservative fluid and mixes acquisition tryptose
The trypsase powder 1.1g is added in every 100mL spermatozoa preservative fluid in enzyme solutions；
The sperm storage formula of liquid are as follows: NaCl 8.5g/L, KCl 0.5g/L, CaCl20.16g/L、MgSO4·7H2O
0.3g/L、Na2HPO4·H2O 0.08g/L、KH2PO40.08g/L、NaHCO30.37g/L, glucose 1.1g/L；The sperm is protected
Liquid storage pH=7.2.
Step 2, liquefacient duration: the mature sperm of white crucian carp is added in above-mentioned trypsin solution and under the conditions of 24 DEG C
Handle 17min；
Step 3, insemination hatching: by the sperm handled by step 2 (from white crucian carp in embodiment 3) and hybridized prussian carp
Mature egg (from " hybridized prussian carp A+ system " in embodiment 3) carries out dry method insemination, and it is different that gained fertilized eggs carry out hatching acquisition
Source octoploid pond crucian carp.The hatching pattern is to be placed in fertilized eggs to hatch in ceramic whiteware disk and change half water in every 12 hours.
For above-mentioned trypsin solution concentration and liquefacient duration time, acquisition is determined by relevant parallel laboratory test.
Too high or too low trypsin solution concentration and too long or too short liquefacient duration time, meeting is found through experiments that in the present invention
Adverse effect brought to survival rate of fish fry and allooctaploid digit rate index simultaneously, and trypsin solution provided by the invention
Concentration and liquefacient duration time are then to help to obtain ideal survival rate of fish fry and allooctaploid digit rate index simultaneously.
Embodiment 7
About Ploidy detection mode:
Step 1, it takes blood: reaching the fry of 1.5cm for length and 0.1 μ L of blood is taken using docking, for reaching six-month-old
Fry use cuts fin and takes 0.1 μ L of blood；
Step 2, it detects: adopted new blood is placed in the (source: moral CyStain DNA1Step that 200 μ L are pre-chilled through 4 DEG C
State Partec) mix in solution after, place on ice, and utilize flow cytometer progress DNA content detection.
The present invention carries out the result of Ploidy detection and the octoploid fish of acquisition to resulting bottle generation is hatched in embodiment 4,5,6
The result of the survival rate of seedling is as follows:
| Group | Embodiment 4 | Embodiment 5 | Embodiment 6 |
| Survival rate | 3.i | 2.8 | 2.4 |
| Allooctaploid digit rate | 16.8 | 16.5 | 16.3 |
Remarks:
Survival rate=(normal fry mantissa/total insemination fish-egg number when starting to ingest) × 100%
Allooctaploid digit rate=(octoploid fry number/total fry mantissa detected) × 100%
Claims (7)
1. a kind of method of efficiently initiative allooctaploid pond crucian carp, it is characterised in that: described manually to award including the artificial insemination stage
The smart stage includes following operating procedure:
Step 1, configure trypsin solution: it is molten that trypsase powder is added to mixing acquisition trypsase in spermatozoa preservative fluid
The trypsase powder 0.9-1.1g is added in every 100mL spermatozoa preservative fluid in liquid；
Step 2, liquefacient duration: the mature sperm of white crucian carp is added in above-mentioned trypsin solution and under the conditions of 22-24 DEG C
Handle 13-17min；
Step 3, the mature egg of the sperm and hybridized prussian carp that handle by step 2 insemination hatching: is subjected to dry method insemination, gained
Fertilized eggs carry out hatching and obtain allooctaploid pond crucian carp.
2. the method for efficiently initiative allooctaploid pond crucian carp as described in claim 1, it is characterised in that: the spermatozoa preservative fluid
Formula are as follows: NaCl 7.5-8.5g/L, KCl 0.3-0.5g/L, CaCl2 0.12-0.16g/L、MgSO4·7H2O 0.1-0.3g/
L、Na2HPO4·H2O 0.04-0.08g/L、KH2PO4 0.04-0.08g/L、NaHCO30.33-0.37g/L, glucose 0.9-
1.1g/L；The spermatozoa preservative fluid pH=7.0-7.2.
3. the method for efficiently initiative allooctaploid pond crucian carp as described in claim 1, it is characterised in that: the hatching pattern is
Fertilized eggs are placed in and hatches in ceramic whiteware disk and changes half water in every 12 hours, or fertilized eggs are placed in be put into water tank in mesh sheet and are oxygenated
Hatching.
4. the method for efficiently initiative allooctaploid pond crucian carp as described in claim 1, it is characterised in that: this method further includes different
Educate pond crucian carp parent culture and reproductive stage, the hybridized prussian carp parent culture and reproductive stage the following steps are included:
Step 1, pond-inputting prepares: in winter, first carrying out dry pool disinfection to the parent culture pool before parent's pond-inputting, kills trash fish, then
Xin Shui is poured into again to the parent culture pool, while isolation net cover is set to prevent trash fish to be mixed into cultivation in parent culture pool water inlet
The pool；
Step 2, parent culture: thoroughly cleaning up the pond before parent's pond-inputting, and carries out male and female screening, eliminates all milters；It will be close after the beginning of spring
This cultivation pool depth of water is improved to 1.5-2.0 meters；
The feeding volume of feed is calculated according to the 2-4% of parent's weight total amount during parent culture；
Splash during parent culture into the parent culture pool each 3000-4500kg/hm of decomposed organic fertilizer2Or the EM of fermentation
1200 milliliter/mu of bacterium solution/7-15 days reduces Dissolved Oxygen in Water to 2-4mg/L；
When parent culture pool water temperature is at 16 DEG C, high water level seine, check parent's maturity, to the development of ovary to fourth phase mid-term with
And parent later hastens parturition immediately, turns to support to interior to do induced reproduction preparation to the parent of the development of ovary to the initial stage fourth phase；
Step 3, it hastens parturition: using+500 international units of 1ug/kg dioxone+1mg/kg pituitary/kg human chorionic gonadotrophin
The mixed liquor of+5ug/kg ovulation induction element carries out injection to parent and hastens parturition, and the parent after injection is hastened parturition is put into 21 DEG C of constant indoor temperature streams
Oviposition is waited in the cylinder of water.
5. the method for efficiently initiative allooctaploid pond crucian carp as described in claim 1, it is characterised in that: this method further includes incubating
The Ploidy detection mode of filial generation after change, the Ploidy detection mode the following steps are included:
Step 1, take blood: the fry for reaching 1.5cm for length takes 0.1 μ L of blood using docking, for the fry for reaching six-month-old
0.1 μ L of blood is taken using fin is cut；
Step 2, it detects: adopted new blood being placed in the CyStain DNA 1Step solution that 200 μ L are pre-chilled through 4 DEG C and is mixed
Afterwards, it places on ice, and carries out DNA content detection using flow cytometer.
6. the method for efficiently initiative allooctaploid pond crucian carp as claimed in claim 4, it is characterised in that: white crucian carp male parent is by above-mentioned
The dosage injection of mixed liquor one third is hastened parturition, and the white crucian carp male parent is 2 more than age healthy and strong individuals, and weight is in 300-500g.
7. the method for efficiently initiative allooctaploid pond crucian carp as claimed in claim 4, it is characterised in that: the parent culture mistake
Cheng Zhong rises sharply area for will appear temperature March, then divides cylinder temporarily to support parent at 16-20 DEG C of temperature and follow for 18-22 DEG C indoors
Constant temperature is raised in ring water body, then carries out subsequent hasten parturition.
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