CN1709047A

CN1709047A - Fish gynogenesis method

Info

Publication number: CN1709047A
Application number: CNA2005100316908A
Authority: CN
Inventors: 刘少军; 刘筠; 周工健; 罗凯坤; 孙远东; 张纯
Original assignee: Hunan Normal University
Current assignee: Hunan Normal University
Priority date: 2005-06-10
Filing date: 2005-06-10
Publication date: 2005-12-21
Anticipated expiration: 2025-06-10
Also published as: CN100382686C

Abstract

The present invention relates to a gynogenesis technique of fishes. Said invention uses diploid ovum of tetraploid crusian carp as material, and uses mirror carp or triangular bream sperm inactivated by UV ray to activate the ovum, the activated ovum can be incubated to obtain fry, said fry is fed and developed into the posterity G1 capable of producing diploid ovum, then adopts similar method to obtain G2 and G3. The incubated female diploid crusian carp system capable of producing diploid ovum is important genetic resource.

Description

The gynogenesis method of a kind of fish

Technical field:

The present invention relates to the cultivation of fish, be specifically related to the gynogenesis technology of fish.

Background technology:

The gynogenesis technology has important effect at fish aspect such as purify aspect breeding, the Sex determination of inquiring into fish and the fish monosex cultivation.Both at home and abroad about the existing many reports of fish gynogenesis research, the research report is all arranged as the gynogenesiss of fishes such as carp, grass carp, silver carp, rainbow trout, Tilapia mossambica, red crucian carp, white bass fish.The cultivation of general gynogenesis fishes is the activation of spermatozoa of employing fish monoploid ovum through the genetic material deactivation, handles the dliploid offspring that the genetic material of the main dependence of formation ovum is grown again through chromosomal doubling.Being used for activating the sperm of ovum, generally is the allos sperm, though allos sperm process inactivation treatment, but have " different smart effect ", the effect that this " different smart effect " may produce the gynogenesis offspring.In fish purification breeding, it is generally acknowledged 1 generation gynogenesis fishes the effect of isozygotying can be equivalent to many effects of purifying for the selfing of fish, can in the gynogenesis offspring, obtain the individuality that genetic character improves by seed selection.In fish gynogenesis process, the ovum of using generally all is the monoploid ovum, the process that the monoploid ovum must have a chromosome doubling to handle in the gynogenesis process, growth has adverse effect and this processing procedure is to ovum, has reduced offspring's survival rate.

Explore a kind of gynogenesis method, have using value with the viability that improves the offspring without the chromosome doubling processing procedure.

Summary of the invention:

The present invention aims to provide a kind of gynogenesis technology of diplont ovum, not only improves offspring's viability, improvement fish proterties, and the diplont ovum resource can also be provided.

The objective of the invention is to be achieved through the following technical solutions:

A kind of fish gynogenesis method, the dliploid mature egg that produces with tetraploid crucian carp carp is a material, with mixing with it through the scattered mirror carp of ultraviolet inactivation or the sperm of megalobrama amblycephala, mix after 1～2 minute, placed hatching under 20～21 ℃ of water temperature conditions by the diplont ovum of activation of spermatozoa, the fry that hatches is cultured in the pond, has obtained to produce the first generation gynogenesis diploid crucian carp carp G of diplont ovum ₁, with gynogenesis diploid crucian carp carp G ₁The diplont ovum that produces is a material, obtains to produce the second generation gynogenesis diploid crucian carp carp G of diplont ovum again through said method ₂, through obtaining G with quadrat method ₃, cultivate the diploid female crucian carp carp strain that produces diplont ovum.

Be described in further detail the present invention below in conjunction with accompanying drawing

Description of drawings:

Fig. 1 the 1st generation gynogenesis diploid crucian carp carp (G ₁) chromosome (2n=100)

Fig. 2 the 2nd generation gynogenesis diploid crucian carp carp (G ₂) chromosome (2n=100)

The original female parent of tetraploid crucian carp carp (4n=200) is red crucian carp (Carassius auratus red var.), and original male parent is Xiangjiang wild carps (Cyprinus carpio).The body colour of tetraploid crucian carp carp is a cinerous, some feature gets involved between carp and the red crucian carp on its profile, (Xiangjiang wild carps has 2 pairs significantly must if any two pairs of very short palpuses, and red crucian carp need not), female, male tetraploid crucian carp carp can produce diplont ovum and diploid sperm respectively, but tetraploid crucian carp carp remains at aspects such as resistance, growth rate to be improved.

The present invention is a starting material with the diplont ovum that tetraploid crucian carp carp produces, with activation of spermatozoa tetraploid crucian carp carp, the G of scattered mirror carp or megalobrama amblycephala ₁And G ₂The diplont ovum that produces carries out gynogenesis.Concrete grammar is: extrude reach sexually matured male scattered mirror carp (Cyprinus carpio var.) or megalobrama amblycephala (Megalobrama amblycephala) seminal fluid in culture dish, use Hank ' s liquid dilution seminal fluid, the ratio of Hank ' s liquid and seminal fluid is 4: 1, the seminal fluid of dilution distributes with thin layer in culture dish, the culture dish of containing seminal fluid is placed on (rotary speed is 30～40 rev/mins) on the shaking table that is lined with ice bag, and place 2 power to be respectively treatment with irradiation under 15 watts the uviol lamp, the distance of uviol lamp and seminal fluid is 10～12 centimetres, and irradiation time is 30 minutes.Hank ' s formula of liquid is as follows: KCL 0.40g, NaCL 8.00g, NaHCO ₃0.35g, KH ₂PO ₄0.06g, Na ₂HPO ₄7H ₂O 0.09g, Na ₂HPO ₄12H ₂O 0.10g, MgSO ₄7H ₂O 0.10g, MgCL ₂6H ₂O0.10g, CaCL ₂0.14g, Glucose 1.00g; Adding distil water is assigned to 1 liter.

The seminal fluid that shone mixes with the mature egg that tetraploid crucian carp carp produces, and fully stir sperm and ovum with clean chicken feather, after 1～2 minute ovum being tiled fills in the culture dish of clear water, and the dispense with dyeing body doubles to handle, and the embryo is hatched under 20～21 ℃ of water temperatures.The fry that hatches is cultured in the pond.By the method, obtain the first generation gynogenesis diploid Goldfish (G of large number of viable ₁); G ₁Specific function with a large amount of generation diplont ovums, the diplont ovum that they produce is under the activation of spermatozoa of the scattered mirror carp of ultraviolet inactivation or megalobrama amblycephala, and the dispense with dyeing body doubles to handle, and has obtained a large amount of second generation gynogenesis diploid crucian carp carp (G ₂); G ₂The specific function that also has a large amount of generation diplont ovums, the diplont ovum that they produce is under the activation of spermatozoa of the scattered mirror carp of ultraviolet inactivation or megalobrama amblycephala, and the dispense with dyeing body doubles to handle, and has obtained a large amount of third generation gynogenesis diploid crucian carp carp (G ₃).The embryonic development situation of observation experiment fish, and the record embryonic development is to the situation of the percentage and the incubation rate of primitive gut.With the direct method of tableting of nephrocyte dliploid Goldfish chromosome number of somatic is detected.The method of the direct method of tableting of nephrocyte is: cultivate the experiment fish after 1～3 day under 18～22 ℃ of water temperatures, the experiment fish is injected PHA 1～3 time, each dosage is 2～8 μ g/ gram body weight, be 12～24 hours blanking time, drew materials preceding 2～6 hours in dissection, injection colchicine, dosage are 2～4 μ g/ gram body weight.Take out nephridial tissue, under physiological saline, shred nephridial tissue, the hypotonic processing of 0.075mol/L KCL, under 20 ℃ of temperature hypotonic 40～60 minutes; Use glacial acetic acid: methyl alcohol (1: 3) is nephrocyte 1～3 time fixedly; On freezing slide, drip sheet; The dyeing of Giemsa dye liquor.

Experiment showed, that 50%～60% gynogenesis diploid crucian carp carp embryo can grow primitive gut, 12%～15% gynogenesis diploid crucian carp carp embryo can form the offspring of surviving by demoulding.The testing result proof gynogenesis diploid Goldfish chromosome number of somatic of the direct method of tableting of nephrocyte is 2n=100, this gynogenesis diploid Goldfish all is female, their age at sexual maturity was generally 2 years, age at sexual maturity (1 year) than its original female parent-female tetraploid crucian carp carp is postponed, but aspect growth rate and resistance, obviously be eager to excel than tetraploid crucian carp carp, the growth rate of gynogenesis diploid crucian carp carp is than tetraploid crucian carp carp fast 20～30%, the most significant multiplication characteristic of gynogenesis diploid crucian carp carp is that they can produce 90～95% diplont ovum, and these diplont ovums can be used for preparing novel tetraploid and triploid fish.As the growth rate of the novel tetraploid crucian carp carp cultivated of the diploid sperm fertilization that utilizes diplont ovum that gynogenesis diploid crucian carp carp produces and the male crucian carp carp of common tetraploid to produce is faster 10～20% than the growth rate of common tetraploid crucian carp carp, and their disease-resistant, anti-adversity ability and fertility all obviously are eager to excel than common tetraploid crucian carp carp.

Prove that by cell and molecular biology experiment the characteristic of dliploid crucian carp carp hybridization fish generation diplont ovum may be relevant with the endoreduplication of reproductive cell.

The gynogenesis diploid crucian carp carp growth speed that the inventive method is cultivated is fast, and premunition is strong, the reproductive capacity height, and can produce Give birth to a large amount of diplont ovums, can omit the processing procedure of chromosome doubling in the gynogenesis of diplont ovum, greatly Reduced the egg development adverse influence, thereby greatly improved the survival rate of gynogenesis individuality. Two times of gynogenesis The formation of body Goldfish system has created a kind of novel dliploid crucian carp carp hybridization fish strain that can produce a large amount of diplont ovums, Provide important diplont ovum resource for preparing novel triploid and tetraploid fish.

Claims

1, a kind of fish gynogenesis method, it is characterized in that the dliploid mature egg that produces with tetraploid crucian carp carp is a material, with mixing with it through the scattered mirror carp of ultraviolet inactivation or the sperm of megalobrama amblycephala, mix after 1～2 minute, to be placed hatching under 20～21 ℃ of water temperature conditions by the diplont ovum of activation of spermatozoa, the fry that hatches is cultured in the pond, has obtained to produce the first generation gynogenesis diploid crucian carp carp G of diplont ovum ₁, with gynogenesis diploid crucian carp carp G ₁The diplont ovum that produces is a material, obtains to produce the second generation gynogenesis diploid crucian carp carp G of diplont ovum again through said method ₂, through obtaining G with quadrat method ₃, cultivate the diploid female crucian carp carp strain that produces diplont ovum.