CN101606501B - Technical method and device of triploid seed production of rainbow trout - Google Patents
Technical method and device of triploid seed production of rainbow trout Download PDFInfo
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- CN101606501B CN101606501B CN2009100895472A CN200910089547A CN101606501B CN 101606501 B CN101606501 B CN 101606501B CN 2009100895472 A CN2009100895472 A CN 2009100895472A CN 200910089547 A CN200910089547 A CN 200910089547A CN 101606501 B CN101606501 B CN 101606501B
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Abstract
The invention relates to a technical method and a device of triploid seed production of rainbow trout. The technical method comprises the following steps: in proper time after sperm-egg binding, carrying out thermal (shock) treatment on oosperm in low-temperature environment in a certain time interval, then restoring the original low-temperature environment and carrying out cooling water incubation; leading more than 80% of oosperms which need to be incubated into diploid oosperm to generate triploid oosperm after thermal shock treatment; and raising the triploid oosperm into triploid offspring seed (larval fish). The implementation of the technical proposal stops the discharging of a secondary polar body of the oosperm, induces the oosperm to produce another type of oosperm which is fused by the egg,the sperm nucleus and the secondary polar body with respective monoploid chromosome and contains triploid chromosome, namely triploid oosperm, and realize industrialization of triploid seed production. Each thermal inducing incubator can produce more than 40,000 of triploid oosperms for one time, and each reproduction period (October to February) can produce more than 2,000,000 oosperms. The inductivity of the triploid is more than 80%.
Description
Technical field
The present invention relates to technical method of triploid seed production of rainbow trout and device, belong to fish chromosome engineering breeding technical field.
Background technology
Since German biologist has found that ray can be controlled the phenomenon of batrachia fertilization cell chromosome quantity, American-European and Japanese ichthyologist has further developed artificial method control fish fertilization cell chromosome behavior, quantity, character and then has bred the technology of new varieties, i.e. chromosome engineering breeding technique.This is a new and high technology that is different from traditional genetic breeding.
Crux of the present invention is that employing heat shock method is that heat treating process stops the discharge of cold water fishes rainbow trout fertilized egg second polar body to induce the generation triploid zygote.At present domestic still do not have can be by comparison technical examples.Remove this exception in the Asia, only Japan implemented the heat shock method and induced the triploid experiment of rainbow trout.Compare with dliploid, the triploid rainbow trout has individual long big characteristics, but Yamato does not eat the custom of big fish, and the specification of edible rainbow trout all is the small fish about 100g, so do not produce triploid needs.Therefore the induced triploid test of Japan's enforcement is bench-scale small test substantially, and experimental scale mostly is tens of grains to thousands of fertilized eggs, and triploid induction rate reaches 80～95%.Device therefor all is laboratory equipments such as box water heater, and experiment purpose mainly is to inquire into chromosome quantity, behavior Changing Pattern from the biology of fishes angle.Compare with Japan, the characteristics of present technique aspect equipment are to have production-scale triploid induction facility, mainly are 28～30 ℃ of constant temperature water supply devices, have the triploid zygote heat shock induction device (the double incubator of doing) of recoil, spilling water, end drain function.Characteristics technically are fertilized egg to be implemented continuous-flow type imbibition, the processing of continuous-flow type heat shock and the hatching of cold flow water work continuously.Characteristics on scale are to implement production-scale triploid induction.This technology is being implemented achievements conversion at present, has tentatively realized industrialization production of hybrid seeds production, also exists hot water-feed outlet pipeline bore problem less than normal on the equipment.
Nearly ten over several years, the domestic triploid induction test report that once had about temperate water fishes such as carp, crucian carp etc., and test method is hydrostatic pressing force method or cold shock method induced triploid, but only is bench-scale small-scale test.Still the report that does not have industrial experimentation and put into production so far.
Summary of the invention
In order to overcome the deficiency of prior art structure, the present invention releases technical method of triploid seed production of rainbow trout and device.The technical problem to be solved in the present invention:
1 stops fertilized egg the 2nd polar body to be discharged, and makes it to generate the fertilized egg that 1 diploid that 1 diploid that contains the egg cell egg nucleus and carry, 1 diploid that the spermatid sperm nucleus carries and egg cell the 2nd polar body carry promptly contains 3 diploids.
2 stop the 2nd polar body to be discharged, and adopt the heat shock method that is suitable for producing in enormous quantities triploid zygote, the method and the technical parameter of research and development heat shock.
3 provide heat shock to produce the apparatus of triploid eyed eggs in batches.
The technical solution adopted for the present invention to solve the technical problems is:
Technical method of triploid seed production of rainbow trout, ovum, smart in conjunction with after opportune moment, in the regular hour interval, the fertilized egg that is in low temperature environment is carried out heat (shock) and handles, and then be returned to original low temperature environment and carry out cold water and hatch; Make and to breed diplontic fertilized egg, after heat shock is handled, generate triploid zygote more than 80%; Breed triploid seed (postlarva) by triploid zygote again.
Three guardian technique parameters of the present invention:
The beginning opportunity that parameter (1), heat (shock) are handled: under 10 ℃ of water temperatures, the 10th minute begins fertilized egg is carried out heat (shock) processing after the enforcement artificial insemination.
Parameter (2), heat shock temperature: 28～30 ℃.
Parameter (3), heat shock time thresholding: 10～12 minutes.
Technical method of triploid seed production of rainbow trout, contain following steps:
Step 1 is carried out ovum artificial collection, semen collection respectively to rainbow trout;
With single salt (NaCl) isotonic solution shower extraction ovum.Per ten thousand ovum need shower 400 ± ml.
The preparation of single salt isotonic solution
Adopt edible salt, making concentration with the conventional soln preparation method is single salt NaCl aqueous solution of 0.97～0.98%.Before adopting ovum, adopt ovum quantity according to plan on the same day, the isotonic solution of making capacity uses for washing ovum at every turn;
Step 3 is implemented artificial insemination, makes ovum, smart in generating fertilized egg;
Step 10, the triploid seed rearing.
The triploid seed production of rainbow trout device: rainbow trout triploid induction device, contain triploid zygote thermal induction equipment, the triploid postlarva hatches equipment,
Triploid zygote thermal induction equipment contains hot constant temperature water supply device, thermal induction incubator (draining incubator at the bottom of the recoil overflow-type);
Hot constant temperature water supply device: water-feed volume 400kg, in establish heating, attemperating unit, and feed water inlet.Hot water injects the thermal induction incubator from the feed water inlet;
Thermal induction incubator (draining incubator at the bottom of the recoil overflow-type): the thermal induction incubator is made of parts such as hatching pail, end discharge outlet water funnel, water injection pipe and net basins; The net basin is to contain the ovum district with top;
The triploid postlarva hatches equipment: place side by side formula recoil water postlarva membrane incubator, flat flow cylinder mould membrane incubation tank;
Placing side by side formula postlarva membrane incubator constitutes with 6 floatingization of square dishes putting into groove by placing side by side groove, the bottom of each dish is that into water sieves, rear wall is water sieve, the bottom of rear wall extends into water fender, water is gone into dish from the recoil of water inlet sieve, flowing through, the water sieve that goes out from rear wall flows out behind the eyed eggs on trying to get to the heart of a matter, flow to downwards again and tries to get to the heart of a matter, and the water inlet sieve recoil through next one dish enters next dish again;
Flat flow cylinder mould membrane incubation tank: this groove is made of the cobble of 2～3 centimetres of the little cylinder mould of tubulose of long 6cm diameter 3cm of incubation tank and tiling bottom land or diameters, eyed eggs is placed on cylinder mould or the cobble, current are flowed through from water inlet behind cylinder mould or cobble and the eyed eggs, discharge from delivery port;
The triploid postlarva is raised equipment:
3 times of body postlarvas of hexagon are raised groove: the groove material is a fiberglass, is hexagon, and the groove upper edge is around thick 1 centimetre an of circle, high 5 centimetres reinforce belt;
Groove is high 80 centimetres, and last bore and end internal diameter are respectively 80 centimetres and 60 centimetres, and groove central authorities are provided with at the bottom of the bushing type pumping equipment, the general tap of water filling piping of spilling water in the draining; The Water injection tap secund; The water that injects in the groove is circulation type.
The technical solution used in the present invention: be the continuous-flow type heat shock induction triploid of implementing batch process, but every batch of heat shock of existing equipment is handled fertilized egg more than 400,000.Can produce the triploid eyed eggs more than 2,000,000 each breeding period (October～February).
The technical characterictic of this scheme is continuous-flow type thermal treatment, has solved that sealing thermal treatment or pressure treatment easily cause anoxic and the problem that is unsuitable for mass disposal fertilized egg.
Innovation of the present invention (with the main difference point of present dliploid seeding technique)
The operation principle of triploid seed production of the present invention:
As shown in Figure 4, in the mature egg cell 20 of fish, contain egg nucleus and 1 second polar body of carrying 1 diploid (n) (the 1st polar body is discharged from before this) of carrying 1 diploid (n).When spermatid enters egg cell 20, under the stimulation of spermatid 21, second polar body is discharged from outside the ovum together with the chromosome of his 1 multiple, the egg nucleus that carries 1 diploid (n) merges mutually with the sperm nucleus that carries 1 diploid (n), generation has the chromosomal dliploid fertilized egg of diploidy number, and this ovum will develop into liploid fish.
After spermatid 21 enters egg cell 20, machine in due course, take suitable artificial measures (the present invention take be heat shock facture 22), stop the discharge of second polar body, will generate the fertilized egg of containing triple chromosome (n, n, n) that is fused into by egg nucleus, sperm nucleus, second polar body three parts is triploid zygote 23, and this ovum will develop into triploid fish.
Beneficial effect of the present invention, the enforcement of the technical program, stoped the discharge of fertilized egg second polar body, induce fertilized egg become by ovum, sperm nucleus and second polar body carry separately that 1 diploid is fused into to contain the chromosomal fertilized egg of triple be triploid zygote, realized the industrialization of triploid seed production.
Every thermal induction incubator can be produced triploid zygote more than 40,000 at every turn, and can produce more than 2,000,000 each breeding period (October～February).Triploid induction rate reaches more than 80%.
Description of drawings
Fig. 1 for the hot constant temperature water supply device of the present invention and the recoil overflow-type at the bottom of draining incubator device schematic diagram.
Fig. 2 places side by side formula postlarva membrane incubator device schematic diagram for the present invention.
Fig. 3 raises the slot device schematic diagram for 3 times of body postlarvas of hexagon of the present invention.
Fig. 4 is a triploid seed production schematic diagram of the present invention.
Fig. 5 is a recoil wave formula flow schematic diagram of the present invention.
Fig. 6 is a single flow cylinder mould membrane incubation tank device schematic diagram of the present invention.
The present invention is further described below in conjunction with drawings and Examples.
Embodiment
Embodiment 1: as shown in Figure 1, the technical solution adopted for the present invention to solve the technical problems is:
Ovum, smart in conjunction with after opportune moment, in the regular hour interval, the fertilized egg that is in low temperature environment is carried out heat (shock) and handles, and then be returned to original low temperature environment and carry out cold water and hatch.Diplontic fertilized egg should be bred, after heat shock is handled, triploid zygote can be generated more than 80%.Breed triploid seed (postlarva) by triploid zygote again.
Three guardian technique parameters of △ technological means
The beginning opportunity that parameter (1), heat (shock) are handled: under 10 ℃ of water temperatures, the 10th minute begins fertilized egg is carried out heat (shock) processing after the enforcement artificial insemination.
Parameter (2), heat shock temperature: 28～30 ℃.
Parameter (3), heat shock time thresholding: 10～12 minutes.
The key equipment of △ technological means:
Hot constant temperature water supply device: see shown in Figure 1.
Hot constant temperature water supply device 2 is the deltiod box container, water capacity 400kg, and casing connects thermal treatment feed pipe 3; In establish electric calorifie installation and automatic temperature-adjusting controller, casing has water injection pipe 1 outward.
The heat sink of electric calorifie installation, heating are fixed on the bottom of casing.
Temperature controller is the commercially available prod.The heating of electric calorifie installation is connected with temperature controller.Its function is to heat shock processor supply heat (28～30 ℃) water.The water yield keeps 400kg in the hot constant temperature water supply device 2, and water temperature is constant between 28～30 ℃.
Water injection pipe 1 constantly injects 10 ℃ cold water, and feed pipe is constantly discharged 28～30 ℃ hot water, and this hot water directly injects the thermal induction incubator.The water yield keeps 400kg in the water-feed, and water temperature keeps 28～30 ℃ of constant temperature, can supply 10 thermal induction incubators simultaneously, simultaneously 400,000 fertilized eggs is carried out heat shock and handles.
Thermal induction incubator (draining incubator at the bottom of the recoil overflow-type) is seen shown in Figure 1.Its primary structure is Plastic Drum 8, Sheng ovum net basin 7, rising pouring water pipe 6, end drainpipe 9.Architectural feature is that used Sheng ovum net basin 7 is commercially available plastic wire basin, and transparent performance is good, can avoid because of crossing the problem that water does not freely cause fertilized egg death by suffocation.
Rising pouring water pipe 6 can make water go into from the device rising pouring, and upwards recoil goes into to contain ovum net basin 7.The function of end drainpipe 9 is after the heat shock processing finishes, the hot water in the device to be discharged within 1 minute,
Contain ovum net basin 7 and can contain fertilized egg 4～50,000.This incubator has the function of fertilized egg being implemented imbibition, heat shock processing and the hatching of cold flow water concurrently.
Place side by side formula postlarva membrane incubator and see Fig. 2:
Constitute by placing side by side groove 50 and putting into 6 square incubation plates 10 of placing side by side groove, the groove rear end is provided with the bushing type drainage system that can implement end draining of end drainpipe 51 and spilling water drainpipe 52 formations.
The bottom of each incubation plate is that into water sieves, and rear wall is water sieve, and the bottom of rear wall extends into water fender 60, water is gone into incubation plate 10 from the recoil of water inlet sieve, flowing through, the water sieve that goes out from rear wall flows out behind the eyed eggs in the dish, flow to downwards again and tries to get to the heart of a matter, and the water inlet sieve of next incubation plate is gone in recoil again.As shown in Figure 5, current recoil wave formula flows.Each incubation plate holds 1.5 ten thousand of eyed eggses, and 6 incubation plates of every groove can hold 90,000 on ovum and carry out the membrane hatching.This facility possesses the function that makes the postlarva incubation of membrane in the eyed eggs, behind the postlarva membrane, can utilize and place side by side groove and carry out opening and raise.
Single flow cylinder mould membrane incubation tank: see Fig. 6
This groove is made of rectangle incubation tank 101 and the tiling cylinder mould 81 of bottom land or cobble (2～3 centimetres of diameters), and the eyed eggs bulk storage is at the gap location of cylinder mould 8 or cobble, and current are flowed through from water inlet 31 behind cylinder mould or cobble and the eyed eggs, discharge from delivery port 91.The effect of cylinder mould or cobble is to separate eyed eggs, avoids ovum collection heap anoxic, also can avoid ovum to be rushed at the discharge outlet place.Every square meter can hold eyed eggs 3～40,000.After postlarva hatches, remove cylinder mould or cobble, incubation tank can be used for the postlarva opening and raises.
3 times of body postlarvas of hexagon are raised groove 13, as shown in Figure 3,
It is fiberglass that 3 times of body postlarvas of hexagon are raised groove 13 materials, is hexagon, and the groove upper edge can guarantee that around thick 1 centimetre an of circle, high 5 centimetres reinforce belt cell body is indeformable, has reduced the nearly 10 centimetres wide mouth of general garden shape groove again along many occupation space.3 times of body postlarvas of two hexagons are raised between the groove and respectively join with one side, have also played and have reduced the effect that takes up room.Omit 10 centimetres wide mouth edge, reduced the cost of material.
Groove is high 80 centimetres, and last bore and end internal diameter are respectively 80 centimetres and 60 centimetres, and central authorities are provided with sleeve pipe 14 in the groove, sleeve pipe 14 is enclosed within outside the drainpipe 15, the draining sieve aperture is arranged at the bottom of sleeve pipe 14, and the water of trench bottom upwards flows by sieve aperture, overflows outside the groove from the suitable for reading of drainpipe 15 again.
The general tap of water filling piping.The Water injection tap secund flows injection water ringwise in groove.Tank water solubilization 0.25m
3, also want draining in the time of water filling, trough inner water is at any time all in the exchange of flowing, and exchange velocity can be regulated by the control water injection rate, and maximum exchange speed is per hour 10 times.Per hour exchange 6 times, every groove can be put postlarva 10,000 tails of cabrage 1 gram in a suitable place to breed, improves 0.3 times of stocking rate and 1 times respectively than the garden shape groove that occupies same spatial area and box-type groove.
The technical solution used in the present invention is the continuous-flow type heat shock induction triploid that can implement to produce in batches, but every batch of heat shock is handled 400,000 in fertilized egg.One of major technique feature of this scheme is continuous-flow type thermal treatment, has solved sealing thermal treatment or pressure treatment and has easily caused because of the dead problem that is unsuitable for mass disposal fertilized egg of anoxic ovum.The 2nd principal character is to implement the imbibition of cold flow water, continuous-flow type heat shock induction triploid and three technology integrated forms of cold flow water hatching to work continuously.The segmentation operation causes between having solved causes the problem of ovum death because of vibrations.
Embodiment 2:
Triploid seed production of rainbow trout technology:
Step 1, ovum artificial collection, semen collection, single salt isotonic solution is washed ovum, artificial insemination.
Step 3 after 10 minutes, is stopped cold water injection, changes to annotate 28～30 ℃ hot water, induces the generation triploid zygote.
Step: hatching accumulated temperature reaches before and after 220 ℃ of d, and eyed eggs is moved into postlarva membrane incubation tank, hatches to postlarva.
Claims (3)
1. technical method of triploid seed production of rainbow trout, its technical characterictic is:
Under 10 ℃ of water temperatures, the 10th minute begins fertilized egg is carried out the heat shock processing after the enforcement artificial insemination;
Heat shock time thresholding: 10～12 minutes, heat shock temperature: 28～30 ℃;
And then be returned to original low temperature environment and carry out cold water hatching; Should breed diplontic fertilized egg, after heat shock is handled, generate triploid zygote more than 80%;
Breed triploid seed postlarva by triploid zygote again;
Contain following steps:
Step 1 is carried out ovum artificial collection, semen collection respectively to female, male rainbow trout;
Step 2, single salt isotonic solution washing ovum;
Step 3, fertilized egg is obtained in artificial insemination;
Step 4 is implemented 10 ℃ of imbibitions of cold flow water 10 minutes to fertilized egg;
Step 5 was implemented 28～30 ℃ of imbibitions of hot-fluid water, heat shock 10 minutes, stoped the 2nd polar body to be discharged, and induced the generation triploid zygote;
Step 6 is implemented 10 ℃ of imbibitions of cold flow water and is implemented to send out eye after 40 minutes and hatch to hatching accumulated temperature and reach 220 and subsist;
Step 7 is implemented 10 ℃ of postlarva membranes hatchings of cold flow water, reaches the 300 postlarva incubation of membrane of subsisting to hatching accumulated temperature;
Step 8 after will hatching postlarva breed swim under flowing water environment, is implemented to open food and is tamed and dociled and raised 30 days, breeds the triploid postlarva.
2. be used to implement the triploid seed production of rainbow trout device of the described method of claim 1, it is characterized in that containing fertilized egg thermal induction equipment, the triploid postlarva hatches equipment,
Fertilized egg thermal induction equipment contains hot constant temperature water supply device, thermal induction incubator;
Hot constant temperature water supply device: water-feed volume 400kg, in establish heating, attemperating unit and feed water inlet;
The thermal induction incubator: the thermal induction incubator is made of hatching pail water funnel, water injection pipe, net basin and end discharge outlet; The net basin is to contain the ovum district with top;
The triploid postlarva equipment of hatching contains places side by side formula postlarva membrane incubation tank, flat flow cylinder mould membrane incubation tank, 3 times of body postlarvas of hexagon and raises groove;
Place side by side formula postlarva membrane incubator: constitute with 6 square incubation plates putting into groove by placing side by side groove, the bottom of each dish is that into water sieves, rear wall is water sieve, the bottom of rear wall extends into water fender, water is gone into dish from the recoil of water inlet sieve, flowing through, the water sieve that goes out from rear wall flows out behind the eyed eggs in the dish, flow to downwards again and tries to get to the heart of a matter, and the water inlet sieve recoil through next one dish enters next dish again;
Flat flow cylinder mould membrane incubation tank: this groove is made of the cobble of 2～3 centimetres of incubation tank and the tiling long little cylinder mould of 6cm diameter 3cm tubulose of bottom land or diameters, the eyed eggs bulk storage is in the gap of cylinder mould or cobble, current are flowed through from water inlet behind cylinder mould or cobble and the eyed eggs, discharge from delivery port;
3 times of body postlarvas of hexagon are raised groove: the groove material is a fiberglass, is hexagon, and the groove upper edge is around thick 1 centimetre an of circle, high 5 centimetres reinforce belt;
Groove is high 80 centimetres, and internal diameter suitable for reading and end internal diameter are respectively 80 centimetres and 60 centimetres, and groove central authorities are provided with pumping equipment at the bottom of the bushing type, the general tap of water filling piping, Water injection tap secund.
3. triploid seed production of rainbow trout device according to claim 2 is characterized in that:
The described heater of establishing in the hot constant temperature water supply device is that electric calorifie installation, described attemperating unit are temperature controller, and hot constant temperature water supply device also is provided with feed pipe;
The described hatching pail of thermal induction incubator is that the Plastic Drum that has discharge outlet of the described end, described net basin are the rising pouring water pipe for Sheng ovum net basin, described water injection pipe, and the rising pouring water pipe makes water go into from the device rising pouring, and upwards recoil goes into to contain ovum net basin; The function of end discharge outlet is a cold or hot water in the quick displacer, guarantees working continuously of heat shock induction and cold flow water hatching;
3 times of body postlarvas of hexagon are raised groove central authorities and are provided with sleeve pipe, and casing pipe sleeve is outside drainpipe, and the bottom of sleeve pipe has sieve aperture to lead in the groove, and the water of trench bottom overflows outside the groove from the suitable for reading of drainpipe by the sieve aperture overflow;
Flat flow cylinder mould membrane incubation tank, by incubation tank and the tiling bottom land long 6cm, diameter 3cm tubular plastic cylinder mould and bushing type drainage equipment constitute.
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