CN109430121A - A kind of breeding method of megalobrama amblycephala - Google Patents
A kind of breeding method of megalobrama amblycephala Download PDFInfo
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- CN109430121A CN109430121A CN201811430359.7A CN201811430359A CN109430121A CN 109430121 A CN109430121 A CN 109430121A CN 201811430359 A CN201811430359 A CN 201811430359A CN 109430121 A CN109430121 A CN 109430121A
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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 present invention discloses a kind of breeding method of megalobrama amblycephala, includes following steps: (a) inactivation of xenogenesis milter sperm: preparing sperm motility and obviously weakens and the xenogenesis milter sperm of not dead inactivation；(b) megalobrama amblycephala Oocyte Activation: make the xenogenesis milter sperm and megalobrama amblycephala ovum fertilization of inactivation, the megalobrama amblycephala ovum activated；(c) the megalobrama amblycephala ovum of diploid induced activation selects (i) or (ii)；(i) cold shock inducible: 0-6min starts the megalobrama amblycephala ovum that cold shock treatment 10-30min is activated in 0-4 DEG C of environment upon activation；(ii) hydrostatic pressing induces: 0-6min starts the megalobrama amblycephala ovum of the Hydrostatic pressure shock processing 1.5-5min activation using 40-50MPa upon activation；(d) the megalobrama amblycephala egg hatching for inducing diploid described in step (c), obtains the megalobrama amblycephala of gynogenesis.
Description
Technical field
The invention belongs to aquatic products fields, are related to a kind of breeding method of megalobrama amblycephala, in particular to a kind of Heterologous Sperm induction
The method of megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis.
Background technique
Megalobrama amblycephala (Megalobrama amblycephala) is also known as blunt snout bream, is phytophage economic fish, has meat
Delicious, the features such as dressed fish is high, feeding habits are wide, growth is fast, premunition is strong[1].It is newest according to United Nations Food and Agricultural Organization (FAO)
Statistical data shows that about 830,000 tons of output in domestic in 2016, be one of main freshwater aquiculture object of China.However, in recent years
Come, megalobrama amblycephala successively occurs that the speed of growth slows down, sex premature, figure are thinning in the breeding process and meat grade decline etc. becomes
Gesture, growth vigor obviously weaken, the threat that wild germ plasm resource is also mixed by excessive fishing and germplasm[2], breeding choosing
Educate work have become there is an urgent need to.For this purpose, we are on the basis of Poyang Lake excellent wild population and "Pujiang No.1" blunt snout bream breeding
On, start within 2009 separation and the purification work of megalobrama amblycephala lower oxygen concentration resistance new lines.2012, through Hypoxia Stress, unexpected mass incident,
Filter out lower oxygen concentration resistance F2For parent.2013, through Hypoxia Stress again, with Quantitative Genetics optimal linear unbiased prediction
(BLUP)。
Analysis and family selective breeding, separation obtain the F of lower oxygen concentration resistance3Generation[3].Compared to "Pujiang No.1" blunt snout bream wild type
" stretching type " gill small pieces, the gill small pieces on the lower oxygen concentration resistance mutant gill filament are in " embedded type ", and in Hypoxia Stress, mutant can
By reducing the matrix between gill small pieces, to expose more gill small pieces to increase Respiratory area, inhaled with improving the gill to oxygen
The ability of receipts[4].But the development to work with artificial breeding, it is possible to occur increasing inbreeding probability, effective population number is continuous
Reduce etc. feelings because accumulation[5].Therefore, it is necessary to pure lines quickly be established by artificial induction's megalobrama amblycephala gynogenesis, for the group of cultivation
Head triangular bream lower oxygen concentration resistance excellent variety building foundation group.
Bibliography:
[1] Li S F, Cai W Q, Zhou B Y.Variation in morphology and biochemical
genetic markers among populations ofblunt snout bream(Megalobrama
Amblycephala) [J] .Aquaculture, 1993,111:117-127.
[2] Li S F, Cai W Q.Genetic improvement of the herbivorous blunt snout
Bream (Megalobrama amblycephala) [J] .Naga, 2003,26 (1): 20-23.
[3] Li Fugui, Zheng Guodong, Wu Chengbin wait megalobrama amblycephala lower oxygen concentration resistance F3Foundation and its growth under low-oxygen environment
Difference [J] aquatic product journal, 2018,42 (02): 236-245.
[4] Wu C B, Liu Z Y, LiF G, et al.Gill remodeling in response to hypoxia
and temperature occurs in the hypoxia sensitive blunt snout bream(Megalobrama
Amblycephala) [J] .Aquaculture, 2017,479-486.
[5] Xu Zhanning, Li Fugui, Zheng Guodong wait the micro- of megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis population genetic variations
Satellite analyzes [J] aquatic product journal, 2017,41 (3): 330-338.
[6] Beijing Lou Yundong fish breeding [M]: Chinese agriculture publishing house, 1999:153-194.
Summary of the invention
Cold shock method is used, mainly herein with the carp that ultraviolet light is irradiated, red crucian carp (Red crucian carp), crucian carp
The sperm of (crucian carp) three kinds of fishes is stimulus, induces megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis；Meanwhile it adopting for the first time
The research of the carp sperm induction megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis of genetic inactivation has been carried out with water purification platen press.It is this time right
Megalobrama amblycephala lower oxygen concentration resistance new lines artificial gynogenesis inductive condition is groped, it is intended to improve and inquire into megalobrama amblycephala lower oxygen concentration resistance new product
It is the abductive approach of artificial gynogenesis, is megalobrama amblycephala fine-variety breeding and sexual control to preferably utilize gynogenesis technology
Work provides scientific basis.
Being irradiated using ultraviolet light (UV) to carp, red crucian carp and crucian carp sperm makes its genetic inactivation, as Heterologous Sperm
Induction source, with megalobrama amblycephala lower oxygen concentration resistance F4It is fertilized for ovum, megalobrama amblycephala lower oxygen concentration resistance F can be induced4Gynogenesis is carried out for ovum.
By cold shock method and hydrostatic platen press, inhibits second polar body discharge respectively, megalobrama amblycephala lower oxygen concentration resistance gynogenesis two has successfully been obtained
Times body fry.Test result is shown: carp sperm induces megalobrama amblycephala after fertilization 3min, and treatment temperature is 0~4 DEG C, the duration
It is 30min, the survival rate of embryonic development to 30h is 31.1 ± 2.18%, and field run plant hatching rate is 4.56 ± 0.02%；Red crucian
Sperm induces megalobrama amblycephala after fertilization 3min, and treatment temperature is 0~4 DEG C, and the duration is 20min, the survival of embryonic development to 30h
Rate is 48 ± 2.1%, and field run plant hatching rate is 12.56 ± 0.6%；Crucian sperm induces megalobrama amblycephala after fertilization 3min, processing temperature
Degree is 0~4 DEG C, and the duration is 20min, and the survival rate of embryonic development to 30h is 11.2 ± 0.5%, and field run plant hatching rate is
2.1 ± 0.2%.And megalobrama amblycephala lower oxygen concentration resistance F4For ovum after the carp sperm fertilization of genetic inactivation 3min, using the quiet of 40MPa
Hydraulic pressure shock processing 3min, the survival rate of embryonic development to 30h is 53.6 ± 3.08%, field run plant hatching rate for 7.28 ±
0.02%.Comparison discovery, identical processing method, different sperm sources, field run plant hatching rate are as follows: red crucian carp > carp > crucian carp；Similarly
Sperm source, different processing methods, hydrostatic platen press are better than cold shock method.
In order to solve the deficiencies in the prior art, this patent first aspect provides a kind of breeding method of megalobrama amblycephala, described
Breeding method includes the following steps:
(a) inactivation of xenogenesis milter sperm:
Sperm motility is prepared obviously to weaken and the xenogenesis milter sperm of not dead inactivation；
(b) megalobrama amblycephala Oocyte Activation:
Make the xenogenesis milter sperm and megalobrama amblycephala ovum fertilization of inactivation, the megalobrama amblycephala ovum activated；
(c) the megalobrama amblycephala ovum of diploid induced activation selects (i) or (ii)；
(i) cold shock inducible:
0-6min starts the megalobrama amblycephala ovum of the activation described in cold shock treatment 10-30min in 0-4 DEG C of environment upon activation
Son；
(ii) hydrostatic pressing induces:
0-6min starts to swash described in the Hydrostatic pressure shock processing preferred 3min of 1.5-5min using 40-50MPa upon activation
Megalobrama amblycephala ovum living；
(d) the megalobrama amblycephala egg hatching for inducing diploid described in step (c), obtains the megalobrama amblycephala of gynogenesis.
In some embodiments, the xenogenesis milter is selected from carp, red crucian carp, crucian carp.
In some embodiments, in step (a), the xenogenesis milter sperm is irradiated with ultraviolet light, arrives sperm motility
Obvious decrease and not dead, stopping irradiation.
In some embodiments, in step (a), the processing step of milter sperm are as follows:
(a-1) milter sperm is squeezed out；
(a-2) Hank ' SShi liquid is added in the milter sperm, obtains the first mixture；It is preferred that the sperm with it is described
The volume ratio of Hank ' SShi liquid is 1:2-4；
(a-3) first mixture is irradiated with ultraviolet light, until observing that sperm motility obviously weakens and not dead, stopping
Irradiation.
In some embodiments, in step (b), the obtaining step of megalobrama amblycephala ovum are as follows:
(b-1) megalobrama amblycephala pectoral fin base portion injection 800-1200IU/kg bw human chorionic gonadtropin and 3-6 μ g/kg bw
Luteinizing hormone-releasing hormone analog；
(b-2) make activity 6-12 hours in water of the megalobrama amblycephala through injecting；
(b-3) it squeezes the megalobrama amblycephala abdomen and obtains the megalobrama amblycephala ovum.
In some embodiments, in step (b), by the xenogenesis milter sperm of inactivation and megalobrama amblycephala ovum in 20-25
It interacts in the water of DEG C pH7.2-8.0.
In some embodiments, in step (i):
0min upon activation, 3min or 6min start the cold shock treatment 10min in 0-4 DEG C of water-bath, and 20min is or, 30min
The megalobrama amblycephala ovum of the activation.
In some embodiments, in step (ii):
0min upon activation, 3min or 6min start described in the hydrostatic pressing processing 3min using 40MPa, 45MPa or 50MPa
The megalobrama amblycephala ovum of activation.
In some embodiments, the hydrostatic pressing operating procedure are as follows: mix megalobrama amblycephala fertilized eggs and the water of the activation
Conjunction is placed in pressure vessel, is applied on pressure vessel by mechanical pressure and is applied to the megalobrama amblycephala fertilized eggs of the activation with water
Add the hydrostatic pressing.
Second aspect of the present invention provides the breeding method of megalobrama amblycephala as described in the first aspect of the invention in the group's of raising head
The hereditary homozygosity of triangular bream germplasm merit gene accelerates the purposes in breeding paces.
Detailed description of the invention
Fig. 1 is that carp sperm induces the brephic survival rate histogram of megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis；
Fig. 2 is the field run plant hatching rate histogram that carp sperm induces megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis；
Fig. 3 is that red crucian induces the brephic survival rate histogram of megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis；
Fig. 4 is the field run plant hatching rate histogram that red crucian induces megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis；
Fig. 5 is that crucian induces the brephic survival rate histogram of megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis；
Fig. 6 is the field run plant hatching rate histogram that crucian induces megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis；
Fig. 7 is that carp sperm induces the brephic survival rate histogram of megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis；
Fig. 8 is the field run plant hatching rate histogram that carp sperm induces megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis.
In Fig. 1-Fig. 6, each horizontal axis time corresponding three columns from left to right successively indicate cold shock treatment 10min,
20min、30min。
In Fig. 7-Fig. 8, each horizontal axis time corresponding three columns from left to right successively indicate hydrostatic pressing processing 40MPa,
45MPa、50MPa。
Fig. 9 is compareing for monoploid deformity fry (above) and normal fry (following figure).
Specific embodiment
Technical solution in order to preferably explain the present invention, is discussed in detail the embodiment of the present invention with reference to the accompanying drawing.With
Lower embodiment should not be construed as further illustrating the present invention to fixation or limitation of the invention.Unless otherwise specified, real
Applying technical characteristic used in example and could alternatively be has equivalent or identity function or effect under the premise of without departing substantially from inventive concept
Other techniques known in the art features.
1 materials and methods
1.1 material
Test is the megalobrama amblycephala lower oxygen concentration resistance F in 3 ages with raun4Generation is document[5]Disclosed F3For the F of megalobrama amblycephala breeding4Generation,
Weight is selected in 1000g or so, figure is good, sexal maturity and well-developed individual, injects using disposable pectoral fin base portion, mixes
It closes and is hastened parturition using human chorionic gonadtropin (HCG) and luteinizing hormone-releasing hormone analog (LHRH-A2), dosage is
1000IUHCG+5 μ gLHRH-A2/kg bw, the raun for having injected oxytocic drug is placed in circular spawning pond, miniflow
Spun lacing swashs, seine harvesting parent population after 10h, obtains ovum using the method for manual compression abdomen；Male megalobrama amblycephala is filled because of sperm
Abundant, without any processing, existing enchashment is used.The milter for inducing gynogenesis is carp (the auspicious carp of good fortune), red crucian carp, crucian carp (wild crucian carp).Test
Material is taken from the Ministry of Agriculture, Shanghai Ocean University megalobrama amblycephala genetic breeding center.
1.2 test method
1.2.1 the irradiation of milter sperm
The clean white sperm (about 2ml) of extrusion is collected into the straight of 4 DEG C of pre-coolings by the abdomen for slowly squeezing mature milter
In the glass culture dish of diameter 12cm, Hank ' SShi liquid (1:3) dilution of 6ml is added, thickness control, will in 0.1~0.2mm or so
Glass culture dish is lain against on the shaking table equipped with ice bag, is subsequently placed in ultraviolet in the closed device with two 15w ultraviolet lamps
Inactivation, irradiation distance are 15cm or so；The vigor of 1 sperm is observed every 5min, until observing that sperm motility obviously weakens
And it is not dead, stop irradiation, adds up irradiation time about 10-20min.It is spare that sperm after irradiation is put in 4 DEG C of refrigerators.
1.2.2 diploid induces
Cold shock inducible: megalobrama amblycephala ovum with 1.2.1 save in the carp that is irradiated in advance by ultraviolet light, red crucian carp and crucian carp sperm
Mixing, water realization fertilization is added at room temperature, and (incubation temperature is 20-25 DEG C, and activation environment is to be aerated tap water or clean
River water pond water, pH7.2-8.0), then start respectively in after fertilization 0min, 3min, 6min cold respectively in 0-4 DEG C of water-bath
Shock processing 10min, 20min, 30min, inhibit the discharge of second polar body, make the chromosome doubling of ovum, every kind of milter 9 examinations
Group is tested, totally 27 test groups (referring to table 1), every group 2 parallel, hatches in different hatching barrels respectively, cleans 1 every 1-2h
Secondary hatching barrel filter screen, until emergence.By calculating the rate of fertilization (development to gastrul stage) and normal emergence rate of each test group,
To determine suitable initial time and processing duration.Survival rate of the record embryonic development to 30h.
Hydrostatic pressing induction: the hydrostatic pressing is by realizing to fertilized eggs aqueous solution, is by applying hydrostatic to aqueous solution
Pressure directly presses fertilized eggs to inhibit the discharge of fertilized eggs second polar body to realize.Megalobrama amblycephala ovum with 1.2.1 save in advance
Water is added at room temperature and realizes that (incubation temperature is 20-25 DEG C, activation for fertilization for the carp sperm mixing being irradiated through ultraviolet light
Environment is aeration tap water or clean river water pond water, pH7.2-8.0), then respectively in after fertilization 0min, 3min, 6min,
The megalobrama amblycephala fertilized eggs of the activation are mixed with aeration tap water and are placed in pressure vessel, fill in pressure vessel with piston
Mouthful, it is then placed in press machine, is pressurized in 15 seconds relevant pressure (40MPa, 45MPa or 50MPa), is handled after 3min 5 seconds again
Pressure release makes the chromosome doubling of ovum, totally 9 test groups (referring to table 2), every group 2 parallel, respectively different to zero in clock
Hatch in hatching barrel, clean 1 hatching barrel filter screen every 1-2h, until emergence.By the fertilization for calculating each test combinations
Rate (development to gastrul stage) and normal emergence rate, to determine suitable initial time and processing pressure.Embryonic development is recorded to 30h
Survival rate.
Positive control (megalobrama amblycephala ♀ × megalobrama amblycephala ♂), negative control (the carp ♂ of megalobrama amblycephala ♀ × irradiated, group are set simultaneously
The crucian carp ♂ of the red crucian carp ♂ of head triangular bream ♀ × irradiated, megalobrama amblycephala ♀ × irradiated, do not make shock processing) and Hybridization Controls (megalobrama amblycephala ♀
× carp ♂, megalobrama amblycephala ♀ × red crucian carp ♂, megalobrama amblycephala ♀ × crucian carp ♂).
1.2.3 rate of fertilization, hatching rate and survival rate statistics
Record ovum sum, the ovum number that is activated, number of emerging and healthy fry number after hatching emergence 1 week, statistics fertilization
Rate, hatching rate and survival rate, rate of fertilization=(the ovum number that is activated/ovum sum) × 100%, hatching rate=(number of emerging/ovum
Sub- sum) × 100%, survival rate=(healthy seedling/ovum sum) × 100%.Field run plant refers to that naked eyes see the normal seedling of the bodily form,
Without deformity.
2 results
2.1 cold shock treatment conditions
During May tests, water temperature is 23 ± 1 DEG C, using the cold shock technique study carp of genetic inactivation, red crucian,
The treatment conditions of crucian sperm induction megalobrama amblycephala lower oxygen concentration resistance new lines artificial gynogenesis offspring.
Table 1 the result shows that, shock temperature is 0~4 DEG C, and the carp sperm of genetic inactivation induces megalobrama amblycephala lower oxygen concentration resistance new product
It is in gynogenesis test, each combined rate of fertilization difference is simultaneously little, and ratio is from 57% to 85%, average out to (66 ± 8) %；
Field run plant hatching rate is from 0 to 4.56%, significant difference.The red crucian carp sperm of genetic inactivation induces megalobrama amblycephala lower oxygen concentration resistance new lines thelykaryon
In development test, each combined rate of fertilization is variant, and ratio is from 11% to 59%, average out to (38 ± 14) %；And field run plant
Hatching rate is from 1.10 to 12.56%, significant difference.The crucian sperm induction megalobrama amblycephala lower oxygen concentration resistance new lines thelykaryon hair of genetic inactivation
In educating, each combined rate of fertilization is variant, and ratio is from 16% to 43%, average out to (26 ± 8) %；And field run plant hatching rate from
0 to 2.1%, significant difference.
The carp sperm of genetic inactivation induces megalobrama amblycephala lower oxygen concentration resistance new lines after fertilization 3min, and treatment temperature is 0~4 DEG C,
Duration is 30min, and the survival rate of embryonic development to 30h is 31.1 ± 2.18%, is significantly higher than other combinations (P=0.05)
(Fig. 1)；Field run plant hatching rate is 4.56 ± 0.02%, the same with the survival rate of embryonic development 30h, and is significantly higher than other
It combines (P=0.01) (Fig. 2).
The red crucian sperm of genetic inactivation induces megalobrama amblycephala lower oxygen concentration resistance new lines after fertilization 3min, and treatment temperature is 0~4
DEG C, the duration is 20min, and the survival rate of embryonic development to 30h is 48 ± 2.1%, is significantly higher than other combinations (P=0.05)
(Fig. 3)；Field run plant hatching rate is 12.56 ± 0.6%, the same with the survival rate of embryonic development 30h, and is significantly higher than other
It combines (P=0.01) (Fig. 4).
The crucian sperm of genetic inactivation induces megalobrama amblycephala lower oxygen concentration resistance new lines after fertilization 3min, and treatment temperature is 0~4 DEG C,
Duration is 20min, and the survival rate of embryonic development to 30h is 11.2 ± 0.5%, is significantly higher than other combinations (P=0.05)
(Fig. 5)；Field run plant hatching rate is 2.1 ± 0.2%, the same with the survival rate of embryonic development 30h, and is significantly higher than other groups
It closes (P=0.01) (Fig. 6).
1 megalobrama amblycephala lower oxygen concentration resistance F of table4For the test result of the most suitable treatment conditions of cold shock
Note: carp group 1-9, red crucian carp group 10-18, crucian carp group 19-27
1.2 hydrostatic pressing treatment conditions
During test, water temperature is 23 ± 1 DEG C, utilizes the artificial thelykaryon of hydrostatic pressing technique study megalobrama amblycephala lower oxygen concentration resistance new lines
The treatment conditions of development.
Table 2 the result shows that, hydrostatic pressing handle the time be 3min, each combined rate of fertilization difference is simultaneously little, ratio from
63% to 86%, average out to (72 ± 7) %；And field run plant hatching rate is from 0 to 5.66%, significant difference.
The carp of genetic inactivation induces megalobrama amblycephala lower oxygen concentration resistance new lines after fertilization 3min, processing pressure 40MPa, when continuing
Between be 3min, the survival rate of embryonic development to 30h is 53.6 ± 3.08%, is significantly higher than other and combines (P=0.05) (Fig. 7)；
Field run plant hatching rate is 7.28 ± 0.02%, the same with the survival rate of embryonic development 30h, and is significantly higher than other combinations (P
=0.01) (Fig. 8).
The test result of the most suitable treatment conditions of 2 megalobrama amblycephala hydrostatic pressing of table
The embryonic development of 2.3 gynogenesis fishes
This time carp, red crucian carp and the crucian carp sperm of test genetic inactivation induce two times of megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis
Body, compared with normal diploid (fertilization of megalobrama amblycephala male and female), the morphological feature in embryonic development each stage has no significant difference, but
Due to the former through after a period of time cold shock or hydrostatic pressing handle, experienced during embryo extreme environment stress, survival
Individual either constitution still grow, degeneration-resistant be superior to normal diploid.This time experimental design Hybridization Controls (megalobrama amblycephala ♀
× carp ♂, megalobrama amblycephala ♀ × red crucian ♂, megalobrama amblycephala ♀ × crucian ♂), the hybrid embryo overwhelming majority can only develop the tail bud phase, only
There is only a few seedling to hatch, most of is lopsided seedling, is died young successively in 1d.Negative control, i.e. irradiated carp has also been devised
Fish, red crucian and crucian sperm and megalobrama amblycephala ovum fertilization but do not make compareing for cold shock treatment or hydrostatic pressing processing, hatches
Miao Jun show monoploid feature: small, eye is small and black, tail is short and is bent, visible cardiocoelom expands, is cardiovascular under anatomical lens
Hypoplasia and thrombosis etc.；Monoploid, which (hatches and comes in 5 days) before the flat trip of diploid gynogenesis fishes, to die young.Such as figure
Shown in 9, upper figure monoploid deformity fry, the normal fry of the following figure.
2.4 check experiment
By the trimestral megalobrama amblycephala lower oxygen concentration resistance parental autocopulation offspring of membrane and the artificial thelykaryon hair of megalobrama amblycephala lower oxygen concentration resistance new lines
Educate be in each 50 tail megalobrama amblycephala of picking with pond raise, design three parallel laboratory test groups.In August 20 days, progress on November 20 weight
Measurement, data statistics the results are shown in Table 3.Gynogenosis system population growth is significantly faster than control inbreeding population, growth speed as seen from the table
Degree improves 30.4%.Illustrate that gynogenesis group has fast-growth character.
The growth traits of 3 megalobrama amblycephala gynogenesis group of table measures
**p<0.01
3 discuss
3.1 fish gynogenesis breedings
Gynogenesis is that inhereditary material derives from maternal modes of reproduction.Artificial gynogenesis is carried out, not only may be used
To obtain gene pure rapidly, merit is made to reach homozygous fixation with most fast speed, sexual control can also be carried out, reached
Cultivate the purpose of unisexuality seed[6].Gynogenesis offspring is exploitation and the Sex Determination Mechanism, parthenogenesis for cultivating new varieties
Etc. fundamental biological knowledges research provide extremely valuable material.But artificial gynogenesis experiment research be primarily present at
Motility rate is lowly and the offspring of gynogenesis is difficult to identify asking for these two aspects.On identification gynogenesis offspring, relatively good side
Method has: hybrid cannot survive, hybrid form can recognize or hybrid can recognize in biochemical molecular level[6], so, we are with remote edge
Gynogenesis offspring is effectively identified in inactivating sperm hybridization, and can be significantly using the gynogenesis that remote edge inactivating sperm induces
Improve its survival rate.
In order to establish megalobrama amblycephala lower oxygen concentration resistance new lines pure lines for further researching and developing, during test, to group
Head triangular bream lower oxygen concentration resistance F4In generation, has carried out gynogenesis research.Using the carp of ultraviolet light irradiation genetic inactivation, red crucian and crucian essence
Son induction inhibits the discharge of megalobrama amblycephala lower oxygen concentration resistance second polar body using hydrostatic platen press or cold shock method respectively, explores and is suitble to group's head
Shock temperature, initial time and the duration of triangular bream lower oxygen concentration resistance new lines ovum and processing pressure.Test result is shown: carp essence
Son induction megalobrama amblycephala after fertilization 3min, treatment temperature are 0~4 DEG C, and the duration is 30min, the survival rate of embryonic development to 30h
It is 31.1 ± 2.18%, field run plant hatching rate is 4.56 ± 0.02%；Red crucian sperm induces megalobrama amblycephala after fertilization 3min, processing
Temperature is 0~4 DEG C, and the duration is 20min, and the survival rate of embryonic development to 30h is 48 ± 2.1%, and field run plant hatching rate is
12.56 ± 0.6%；Crucian sperm induces megalobrama amblycephala after fertilization 3min, and treatment temperature is 0~4 DEG C, and the duration is 20min, embryo
The survival rate that fetal hair educates 30h is 11.2 ± 0.5%, and field run plant hatching rate is 2.1 ± 0.2%.And megalobrama amblycephala lower oxygen concentration resistance F4Generation
Ovum 3min after the carp sperm fertilization of genetic inactivation handles 3min using the Hydrostatic pressure shock of 40MPa, and embryonic development is arrived
The survival rate of 30h is 53.6 ± 3.08%, and field run plant hatching rate is 7.28 ± 0.02%.Gynogenesis megalobrama amblycephala lower oxygen concentration resistance new product
The acquisition of system, has not only obtained the megalobrama amblycephala of high-purity, but also opens the new way of production unisexuality megalobrama amblycephala.
Since the inhereditary material of gynogenesis group is from female parent, gene expression reaches unanimity, the heredity with height
Homogeney, thus select the high individual of homozygosity from gynogenesis group and carry out second of gynogenesis means, it is sent out for thelykaryon
It educates the breeding of megalobrama amblycephala improved variety and genetic research provides effective way, to establish better breeding material.Meanwhile in thelykaryon
Purposive selection and hybridization are carried out in development group, it is possible to further increase the germplasm of megalobrama amblycephala lower oxygen concentration resistance new lines
Energy.
The effect of 3.2 two methods induction megalobrama amblycephala gynogenesis
One of induction successful key of gynogenesis diploid is the method for the diplodization of ovum chromosome.In this research
In, megalobrama amblycephala lower oxygen concentration resistance new lines gynogenesis is induced using cold shock method, as the result is shown: after fertilization 3min, treatment temperature
It is 0~4 DEG C, duration 30min, the survival rate of embryonic development to 30h is 31.1 ± 2.18%, and field run plant hatching rate is
4.56 ± 0.02%.And hydrostatic pressing is utilized to induce megalobrama amblycephala gynogenesis, the results show that the carp sperm fertilization through genetic inactivation
After fertilization 3min afterwards, processing pressure 40MPa, duration 3min, the survival rate of embryonic development to 30h is 53.6 ±
3.08%, field run plant hatching rate is 7.28 ± 0.02%.Comparison discovery, though two test groups of hydrostatic pressing and cold shock inducible are fertilized
Rate difference is little, but hydrostatic pressing induction group hatching rate and survival rate (53.6%, 7.28%) are respectively than cold shock inducible group
(31.1%, 4.56%) high 22.5% and 2.72%.Illustrate that hydrostatic platen press is not only more easy to operate than cold shock method, effect is also more
Stablize.
The detection of the carp sperm induction megalobrama amblycephala gynogenesis offspring of 3.3 genetic inactivations
This time carp, red crucian carp and crucian carp sperm of the research using irradiating through ultraviolet light carry out to induce megalobrama amblycephala lower oxygen concentration resistance new lines
Gynogenesis.The chromosome number of carp, red crucian carp and crucian carp is all 2n=100, and megalobrama amblycephala is 2n=48, they belong to different Asias
Section does not see megalobrama amblycephala ♀ × carp ♂, megalobrama amblycephala ♀ × red crucian ♂, and megalobrama amblycephala ♀ × crucian ♂ hybridization has the report of offspring, miscellaneous
It hands over control group (megalobrama amblycephala ♀ × carp ♂, megalobrama amblycephala ♀ × red crucian ♂, megalobrama amblycephala ♀ × crucian ♂) also to find no normal fry to incubate
Out.Therefore, it by the carp of ultraviolet light irradiation inactivation, red crucian and crucian sperm, induces megalobrama amblycephala lower oxygen concentration resistance new lines and carries out
The fry that gynogenesis is hatched (cannot normally survive) other than monoploid and lopsided seedling, all should be that normal diploid is female
Caryogenesis fish does not need to carry out special detection.
Above each embodiment is only intended to further illustrate the present invention, is not for limiting protection model of the invention
It encloses, it is all obviously to change based on equivalents made by design of the invention and to each technical solution of the invention
Into each falling within protection scope of the present invention.
Claims (10)
1. a kind of breeding method of megalobrama amblycephala, the breeding method include the following steps:
(a) inactivation of xenogenesis milter sperm:
Sperm motility is prepared obviously to weaken and the xenogenesis milter sperm of not dead inactivation；
(b) megalobrama amblycephala Oocyte Activation:
Make the xenogenesis milter sperm and megalobrama amblycephala ovum fertilization of inactivation, the megalobrama amblycephala ovum activated；
(c) the megalobrama amblycephala ovum of diploid induced activation selects (i) or (ii)；
(i) cold shock inducible:
0-6min starts the megalobrama amblycephala ovum of the activation described in cold shock treatment 10-30min in 0-4 DEG C of environment upon activation；
(ii) hydrostatic pressing induces:
0-6min starts activation described in the Hydrostatic pressure shock processing preferred 3min of 1.5-5min using 40-50MPa upon activation
Megalobrama amblycephala ovum；
(d) the megalobrama amblycephala egg hatching for inducing diploid described in step (c), obtains the megalobrama amblycephala of gynogenesis.
2. the breeding method of megalobrama amblycephala as described in claim 1, it is characterised in that:
The xenogenesis milter is selected from carp, red crucian carp, crucian carp.
3. the breeding method of megalobrama amblycephala as described in claim 1, it is characterised in that:
In step (a), the xenogenesis milter sperm is irradiated with ultraviolet light, obviously weakens to sperm motility and not dead, stops shining
It penetrates.
4. the breeding method of megalobrama amblycephala as claimed in claim 1 or 3, it is characterised in that:
In step (a), the processing step of milter sperm are as follows:
(a-1) milter sperm is squeezed out；
(a-2) Hank ' SShi liquid is added in the milter sperm, obtains the first mixture；It is preferred that the sperm and the Hank ' S
The volume ratio of family name's liquid is 1:2-4；
(a-3) first mixture is irradiated with ultraviolet light, until observing that sperm motility obviously weakens and not dead, stops photograph
It penetrates.
5. the breeding method of megalobrama amblycephala as described in claim 1, it is characterised in that:
In step (b), the obtaining step of megalobrama amblycephala ovum are as follows:
(b-1) megalobrama amblycephala pectoral fin base portion injection 800-1200IU/kgbw human chorionic gonadtropin and 3-6 μ g/kgbw promote corpus luteum
Generate hormone-releasing hormone analog；
(b-2) make activity 6-12 hours in water of the megalobrama amblycephala through injecting；
(b-3) it squeezes the megalobrama amblycephala abdomen and obtains the megalobrama amblycephala ovum.
6. the breeding method of megalobrama amblycephala as described in claim 1, it is characterised in that:
In step (b), in the water of 20-25 DEG C of pH7.2-8.0 mutually by the xenogenesis milter sperm of inactivation and megalobrama amblycephala ovum
Effect.
7. the breeding method of megalobrama amblycephala as described in claim 1, it is characterised in that:
In step (i):
0min upon activation, 3min or 6min start the cold shock treatment 10min in 0-4 DEG C of water-bath, and 20min is or, described in 30min
The megalobrama amblycephala ovum of activation.
8. the breeding method of megalobrama amblycephala as described in claim 1, it is characterised in that:
In step (ii):
0min upon activation, 3min or 6min start to activate described in the hydrostatic pressing processing 3min using 40MPa, 45MPa or 50MPa
Megalobrama amblycephala ovum.
9. the breeding method of megalobrama amblycephala as claimed in claim 1 or 8, it is characterised in that:
The hydrostatic pressing operating procedure are as follows: the megalobrama amblycephala fertilized eggs of the activation are mixed with water and are placed in pressure vessel, are led to
It crosses mechanical pressure and is applied on pressure vessel and the hydrostatic pressing is applied to the megalobrama amblycephala fertilized eggs of the activation and water.
10. the breeding method of megalobrama amblycephala as claimed in any one of claims 1-9 wherein is improving megalobrama amblycephala germplasm merit base
The hereditary homozygosity of cause accelerates the purposes in breeding paces.
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