CN101390503B - Method for establishing super-male yellow cartfish breeding population by copulating super-male fish with physiology spawner - Google Patents
Method for establishing super-male yellow cartfish breeding population by copulating super-male fish with physiology spawner Download PDFInfo
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Abstract
Disclosed is a method for super-male fish to mate physiological spawner to build super-male yellow catfish breeding groups, belonging to the fish physiological genetics; the method belongs to the technical field of controlling the sex of fish through artificial sex reversal, screening, test cross and group expanding. The technical problem to be solved is that issues are the super-male fish mates the sex-reversed physiological spawner; the gynogenesis process is eliminated; through the mating between the sex-reversed physiological spawner and the super-male fish or male fish, and the combination of the sex ratio analysis and the test cross technology, the breeding time is shortened. The invention adopts the following technical proposal: artificial spawning inducement and fertilization; sexreversal, namely, the male fish is sex-reversed into physiological spawner; test cross sex reversal, namely, the physiological spawner and the super-male or ordinary male fish are test-crossed, and the physiological spawner and the super-male fish are selected, and the test cross sex reversal comprises the artificial spawning inducement and fertilization; sex ratio analysis, namely, the sex of the candidate fish is confirmed; offspring determination, screening and identification; group expanding, that is, the breeding groups of the super-male fish and the super-female physiological spawner are expanded; and the breeding production of all-male fish, namely, ordinary spawner mates the super-male fish to produce the all-male fish. The method is used for the breeding and mass production of super-male yellow catfish breeding groups.
Description
Technical field
The method that male yellow cartfish breeding population is set up in supermale fish and the mating of physiology raun belongs to the method that a kind of fish physiological inheritting is bred, and is with technology such as artificial sex reversal, screening, test cross and expansion group control fish sex technical field.
Background technology
Production unisexuality hero has several method, and available (Aquaculture2001 is 197:283-301) as hand picking, the direct inductivity reverse of androgenic hormone, androgenesis, interspecific cross, artificial induction triploid and genetic method etc.But, and can not accurately distinguish female milter because of the labour intensity of hand picking is big.Reverse with the direct inductivity of sex hormone, the danger of sexual hormone residues, many countries forbid already.Serious technology barrier such as then there is inductivity in androgenesis, survival rate is low and YY ♀ physiology raun fertility is extremely low only rests on the experimental stage, does not still have successful example.And three times of artificial inductions not can not accomplish 100% unisexuality, have only Tilapia mossambica interspecific cross and genetic method to achieve success, and apply on producing.But interspecific cross is only limited to several hybrid combinations that Tilapia mossambica belongs to, and spawning rate is lower.The genetics technology that only is called GMT is applicable to the fish of male gamete abnormal shape, and method is reliable, stable, and environmentally friendly, without any adverse effects, can produce full milter in a large number.As far back as last century five, the sixties, Japan Yamamoto (Genetics40:406-419, Exp.Zool.137:227-262, Genetics55:329-336) when blue or green Medaka Sex determination of research and sex-linked inheritance, utilize female sex hormone that XY ♂ milter is reversed into XY ♀ physiology raun, and with the mating of XY ♂ milter, obtain YY ♂ supermale fish first, proof YY ♂ supermale fish can survive, and can feminize, become YY ♂ physiology superfemale fish, YY ♀ physiology superfemale fish and the mating of YY ♂ supermale fish just can produce YY ♂ supermale raun in a large number.1980, China Yang Yong select (Acta Genetica Sinica, 1980,7 (3): 241-246) wait application three series mating approach to produce hereditary complete male Mozambique Tilapia mossambica and succeed, basically be exactly the method for the reverse of usability hormonal and offspring's test cross, obtain YY ♂ supermale fish; 1989, Varadaraj etc. also proposed similar method and obtain YY ♂ supermale Mozambique Tilapia mossambica.After this, Mair etc. 1988, Baroillerant Jalabit 1989, Scoff etc. also delivered relevant research paper in 1989 in succession and propose identical method, obtain YY ♂ supermale bolti (Ph.D.thesis.University of maies.Swansea, Aquat.Living Resour.2:105-116, Aquaculture, 78:235-257) on the basis of these work, the breeding plan that Mair (Aquat.Sci.54:396-404) etc. reported the complete male bolti of large-scale production in 1997, their result of study, just so-called full milter Genetic Manipulative Technology (
).This technology is to obtain YY ♂ supermale fish and YY ♀ physiology superfemale fish at last by the hormonal reverse of five generations half and offspring's test cross, and produces the full milter of XY ♂ by YY ♂ supermale fish and the common raun of XX ♀.Adopt the breeding cycle of this technology longer, and, prevent to mix, could achieve success at last by for the necessary strict control of the test fish of test cross.As seen, for generation cycle long fish, just may not be first-selected method, must employing other effective technical means comparatively.1989, Varadaraj.K. (Current ScienceVol.58, NO, 8:434-441) grade has reported that hormonal reverses the Tilapia mossambica in conjunction with gynogenesis technology generation supermale Mozambique, this technology it is said and the output of YY ♂ supermale fish can be brought up to 50% from 25%, and can shorten 22～8 months breeding time, and the scheme of the full milter of commercial large-scale production is proposed.But do not see the report of on producing, applying.There is no report as for the sex reversal of yellow cartfish and gynogenesis and YY ♂ supermale fish, full milter.Aquatic institute of the Chinese Academy of Sciences had once applied for patent (application number 200610166518.8, publication number: CN1994027A) in 2006.12.28 with regard to " sex reversal and gynogenesis continue to produce complete male yellow cartfish method ".Liu Hanqin etc. mention the integrated technology that adopts sex reversal to combine with gynogenesis first, set up the breeding of method (aquatile journal, 2007,31 (5): 98-101) that continue to produce supermale yellow cartfish YY ♂ and complete male yellow cartfish XY ♂.It is compared with external GMT genetics technology, can shorten two generations of breeding cycle, improves about 1 times of supermale fish output capacity.
From the above file of quoting as can be known: some kinds of methods having reported production unisexuality milter; Utilize female sex hormone that milter is reversed into the physiology raun, can obtain the supermale fish through mating, test cross; Utilize full milter Genetic Manipulative Technology (GMT), reverse and offspring's test cross obtains YY ♂ supermale fish and YY ♀ physiology superfemale fish at last, and can produce the full milter of XY ♂ through the hormonal in some generations; The method that utilization sex reversal and gynogenesis continue to produce complete male yellow cartfish is disclosed.These innovation and creation have promoted the development of breeding of fish cell engineering and artificial sex controlling technology.But also have following problem simultaneously: the gynogenesis cold shock is handled has damage to genetic material; The survival rate of two letter body gynogenesis fishes is undesirable; Large-scale production waits to improve; Breeding cycle has to be shortened.
Summary of the invention
Technical problem to be solved by this invention is: 1. adopt the XY ♀ physiology raun mating of YY ♂ supermale yellow cartfish and sex reversal, improve the output of YY ♂ supermale fish.2. save the gynogenesis operation, reduce of the infringement of artificial induction's gynogenesis, improve the pick-up rate of YY ♂ supermale fish the fertilized egg genetic material.3. usability reverses XY ♀ physiology raun combines technological means such as offspring's test cross, the analysis of property ratio with YY ♂ supermale fish or the mating of XY ♂ milter combination shortening breeding time.
For this reason, the technical solution used in the present invention is a kind of method, and it is undertaken by following operation:
1. artificial induced spawning and artificial insemination: getting body surface does not have that wound, belly are expanded, soft common female yellow cartfish, uses ocyodinic HCG and LRH-A
2Every fish pectoral fin base portion is carried out a shot, and dosage is HCG800～1000IU+LRH-A
25～8 μ g/ tails are placed in 26 ℃ the water, can extrude ripe ovum after 24～28 hours, place on the ice bath low temperature to preserve standby it; Choose gonapophysis little red milter HCG injection and LRH-A
2The ocyodinic that reduces by half is dissected it and to be taken out spermary, with scissors it is shredded, and preserves the liquid filter wash with sperm again and goes out sperm, places on the ice bath low temperature to preserve standby it; Getting ovum mixes with sperm and carries out the dry method artificial insemination; Place the fertilization ovum 27 ℃ of biochemical incubator constant temperature to hatch into milter and raun fry and whole membrane; After treating absorption of yolk at its, fry uses artemia larvae as opening erbium material.
2. sex reversal: common male yellow cartfish sex reversal is become the physiology raun, with artemia larvae as the oestrogenic hormone carrier, it was soaked 1 hour in the 17-α of 100～200 μ g/L ethinyl estradiol solution, suction filtration takes out the estrogenic halogen worm of the enrichment artificial propagation yellow cartfish seedling of initial feeding of throwing something and feeding again, every morning, afternoon and evening respectively throw something and feed once, feeding volume is as the criterion to eat up in 30 minutes, threw something and fed continuously 40 days, about 50% XY ♂ milter sex reversal becomes XY ♀ physiology raun in the fry that survives after female sex hormone is handled, XX ♀ raun is constant, with the juvenile fish of sex reversal be transferred to cement pit with in the soft-shelled turtle feed support to sexual maturity.
3. test cross and sex reversal: with XY ♀ physiology raun and YY ♂ supermale fish or the common milter test cross of XY ♂, screening XY ♀ physiology raun
3.1 artificial induced spawning: get sexually matured candidate XY ♀ physiology raun and common raun and carry out conventional artificial induced spawning.
3.2 artificial insemination: the female fish egg of every tail is divided into two parts, a and XY ♂ milter sperm insemination, another part inseminated with YY ♂ supermale milt.
3.3 property is than analyzing: candidate supermale fish and physiology superfemale fish are standby hatch fry with sex reversal fertilization ovum after, the fry of each mating combination is divided into two parts again, support temporarily in net cage respectively, get wherein 1/3rd fries with behind artemia larvae and the soft-shelled turtle forage feed to three month, take out 30～50 tail juvenile fish by tail lumbar injection glacial acetic acid fixer, just can differentiate spermary or ovary from the sexual gland form after the dissection, analyze sex ratio; Remaining juvenile fish moves in the cement pit foster standby as the YY ♂ supermale fish of candidate to sexual maturity.2/3rds fry in addition, the method for using the oestrogenic hormone inductivity to reverse moves into after 40 days through HORMONE TREATMENT again and to support sexual maturity in the cement pit, and is standby as the YY ♀ physiology superfemale fish of candidate.
3.4 other affirmation of candidate piscinity
3.4.1XY test cross of ♀ physiology raun and the affirmation of property than analysis and XY ♀ physiology raun, with XY ♀ physiology raun and the common milter mating of XY ♂, produce the common raun of 25%XX ♀, 50%XY ♂ milter and 25%YY ♂ supermale fish, the property than analyzing the result, confirm XY ♀ physiology raun, i.e. XY ♀ * XY ♂ → 25%XX ♀+50%XY ♂+25%YY ♂.
3.4.2XX offspring's test cross of the common raun of ♀, property ratio are analyzed and the affirmation of the common raun of XX ♀: with common raun of XX ♀ and the common milter mating of XY ♂, its offspring's male and female fish respectively is 50%, can confirm than analyzing whether tested fish is the common raun of XX ♀ according to property, be XX ♀ * XY ♂ → 50%XX ♀+50%XY ♂, the sex ratios that compare two mating combinations, just can determine the genetic sex of candidate fish, it is standby to filter out XY ♀ physiology raun, and what be confirmed to be the common raun of XX ♀ abandons it.
4. test cross, screening, evaluation YY ♂ supermale fish and YY ♀ physiology superfemale fish
4.1YY the mensuration of ♂ supermale fish: get operation 3.3 sexually matured YY ♂ supermale fishes and the common raun mating of XX ♀,, can confirm that then tested fish is a YY ♂ supermale fish as XX ♀ * YY ♂ → 100%XY ♂; As XX ♀ * XY ♂ → 50%XX ♀+50%XY ♂, can confirm that then tested fish is the common milter of XY ♂.
4.2YY the test cross of ♀ physiology superfemale fish: get operation 3.3 sexually matured candidate YY ♀ physiology superfemale fish and the common milter mating of XY ♂, then YY ♀ * XY ♂ → 50%XY ♂+50%YY ♂ promptly produces 100% milter; And the common milter mating of XY ♀ physiology raun and XY ♂, then XY ♀ * XY ♂ → 25%XX ♀+50%XY ♂+25%YY ♂ promptly produces common raun of 25%XX ♀ and 75%XY ♂ milter.
4.3 screening, identify: than analyzing, can identify the genetic sex of candidate tested fish according to two mating combination offspring test crosses, filter out YY ♂ supermale fish and YY ♀ physiology superfemale fish is standby.
5. expand the group: the quick expansion group of YY ♂ supermale fish and YY ♀ physiology superfemale fish breeding population, get YY ♂ supermale fish and the mating of YY ♀ physiology superfemale fish that operation 4 has been identified, produce 100%YY ♂ supermale fish, when the fry opening, get a part of YY ♂ supermale fry by operation 2, become YY ♀ physiology superfemale fish with the reverse of oestrogenic hormone inductivity, promptly
The physiology superfemale fish.
6. the full milter production of hybrid seeds: with common raun of XX ♀ and the mating of YY supermale fish, the complete male yellow cartfish of the XY ♂ that then is produced on a large scale, i.e. XX ♀ * complete male yellow cartfish of YY ♂ → 100%XY ♂.
This invention has following beneficial effect compared with prior art:
The first, adopt the XY ♀ physiology raun mating of YY ♂ supermale yellow cartfish and sex reversal, the output of the YY ♂ supermale fish of generation 50% and the 25%YY ♂ supermale fish that produced with XY ♀ physiology raun and the mating of XY ♂ milter is compared and is doubled.
The second, because of having saved the gynogenesis operation, can reduce of the damage of artificial induction's gynogenesis to the fertilized egg genetic material, improved the pick-up rate of YY ♂ supermale fish.
The 3rd, owing to adopted XY ♂ physiology raun and YY ♂ supermale fish or the common milter mating of XY ♂ method, can obtain a large amount of YY ♂ supermale fishes rapidly, product can carry out in batches.And improve the genetic diversity of supermale fish, be one of long-term many technological means for sustainable utilization of supermale fish.
The 4th, also utilized sex reversal XY ♀ physiology raun and YY ♂ supermale fish or the common milter mating of XY ♂ just and combined offspring's test cross than analyzing, make screening XY ♀ physiology raun, YY ♂ supermale fish, YY ♀ physiology superfemale fish process, only needed for 3 generations just can set up the breeding population of supermale yellow cartfish, it is applicable to the foundation of the breeding of method of the special-shaped fish supermale of male gamete fish, full milter.This technology than external GMT technology shortened two generation half breeding time; Compare in conjunction with the gynogenesis technology with sex reversal, then saved half for the time, as seen, the method for this breeding population is fast, effectively.
In view of this, the present invention is with a wide range of applications.
Description of drawings
Accompanying drawing is the process flow block diagram of " method that super yellow cartfish breeding population is set up in supermale fish and the mating of physiology raun ".It is made up of following each several part block diagram:
1. artificial induced spawning and artificial insemination
2. sex reversal
3. test cross and sex reversal
4. test cross, screening, evaluation
5. expand the group
6. the full milter production of hybrid seeds
Solid line between the block diagram and dotted line have equal function, but are not communicated with mutually between them.
Embodiment
It is to realize optimal way of the present invention that the applicant thinks with reference to operation shown in the drawings.The particular content of these operations is as follows:
1. artificial induced spawning and artificial insemination: according to experiment or need of industrial production, the body surface of getting some quantity does not have that wound, belly are expanded, soft common female yellow cartfish, carry out a shot for every fish pectoral fin base portion, the ocyodinic and the dosage of use are as follows: HCG900IU+LRH-A
26 μ g/ tails, ocyodinic HCG and LRH-A
2Be the product of Hangzhou animal drug factory and the Xiaoshan first veterinary drug Manufacturing Co., Ltd.Fish is placed 26 ℃ water, after about 26 hours, can extrude ripe ovum, place on the ice bath low temperature to preserve standby it.Choose the same ocyodinic that the little red milter injection of gonapophysis reduces by half simultaneously, because of its spermary structure is dendroid, seminal fluid can not squeezed in the back of hastening parturition, and needs to dissect the taking-up spermary, shred the back with scissors and go out sperm, still place the molten low temperature of going up of ice to preserve standby with sperm preservation liquid filter wash.Hundred to thousand ovums of peeking as required mix with sperm and carry out the dry method artificial insemination, and fertilized egg constant temperature in 27 ℃ of biochemical incubators is hatched to the whole membranes of fry.After treating absorption of yolk at its, fry uses artemia larvae as opening erbium material.
2. sex reversal: common male yellow cartfish sex reversal is become the physiology raun,, that it is first at the 17-α of 150 μ g/L ethinyl estradiol (EE with artemia larvae as the oestrogenic hormone carrier
2Sigma) soaked in the solution 1 hour, suction filtration takes out the estrogenic halogen worm of the enrichment artificial propagation yellow cartfish seedling of initial feeding of throwing something and feeding again, and every morning, afternoon and evening respectively throw something and feed once, and feeding volume is as the criterion to eat up in 30 minutes.Threw something and fed 40 days continuously, experimental results show that survival rate is more than 60%, the sex reversal rate reaches more than 95%, because yellow cartfish is the fish of male gamete abnormal shape, its sex system is XX ♀/XY ♂, and the ratio of female milter is approximately 1: 1.So, in the fry that after sex hormone is handled, survives, have 50% to be that the sex reversal of XY ♂ milter becomes XY ♀ physiology raun approximately, itself and juvenile fish after hormonal reverses are transferred in the cement pit in the lump, be that 43% soft-shelled turtle feed is foster to sexual maturity with protein content.
3. test cross and sex reversal: with XY ♀ physiology raun and YY ♂ supermale fish or the common milter test cross of XY ♂, screening XY ♀ physiology raun.Can be divided into following steps carries out:
3.1 artificial induced spawning: get sexually matured candidate XY ♀ physiology raun (comprising XX ♀ raun), carry out artificial induced spawning by conventional method described in the operation 1.
3.2 artificial insemination: the female fish-egg of every tail is divided into two parts, a ovum and the insemination of XY ♂ milter sperm; Another part ovum and the insemination of YY ♂ supermale milt.
3.3 property is continued to employ and sex reversal than analysis, candidate supermale (female) fish: after fertilized egg is hatched fry, the fry of each mating combination is divided into two parts again, support temporarily respectively in 1 square metre net cage, get wherein 1/3rd fries with artemia larvae with behind the soft-shelled turtle forage feed to three month, take out 30～50 tail juvenile fish by tail lumbar injection glacial acetic acid fixer, can accurately differentiate spermary or ovary after the dissection, analyze sex ratio from the sexual gland form.Remaining juvenile fish moves on in the cement pit foster standby as the YY ♂ supermale fish of candidate to sexual maturity; The method that other 2/3rds fry uses the oestrogenic hormone inductivity to reverse after oestrogenic hormone is handled 40 days, moves into and supports sexual maturity in the cement pit, and is standby as the YY ♀ physiology superfemale fish of candidate.
3.4 other affirmation of candidate piscinity
3.4.1XY the test cross of ♀ physiology raun is analyzed the affirmation of XY ♀ physiology raun with the property ratio: the common milter mating of XY ♀ physiology raun and XY ♂, because chromosome (gene) reorganization produces 25%XX ♀ common raun, 50%XY ♂ milter and 25%YY ♂ supermale fish.Than analyzing the result, confirm XY ♀ physiology raun, i.e. XY ♀ * XY ♂ → 25%XX ♀+50%XY ♂+25%YY ♂ according to property.
3.4.2XX the test cross of the common raun of ♀ is analyzed the affirmation of the common raun of XX ♀ with the property ratio: with common raun of XX ♀ and the common milter mating of XY ♂, because yellow cartfish is the fish of male gamete abnormal shape, its offspring's male and female fish respectively is 50%.Can confirm than analyzing the result whether tested fish is the common raun of XX ♀ according to offspring's property, i.e. XX ♀ * XY ♂ → 50%XX ♀+50%XY ♂.
The sex ratio that makes up of two kinds of mating of 3.4.1 and 3.4.2 relatively can be determined the genetic sex of candidate fish, and it is standby to filter out XY ♀ physiology raun, and what be confirmed to be the common raun of XX ♀ abandons it.
4. measure, screen, identify YY ♂ supermale fish and YY ♀ physiology superfemale fish
4.1YY the mensuration of ♂ supermale fish: get sexually matured candidate YY ♂ supermale fish and the common raun mating of XX ♀ in the operation 3.3, should produce the common milter of 100%XY ♂, as XX ♀ * YY ♂ → 100%XY ♂, can confirm as YY ♂ supermale fish, as XX ♀ * XY ♂ → 50%XX ♀+50%XY ♂, can confirm as the common milter of XY ♂.
4.2YY the test cross of ♀ physiology superfemale fish: get sexually matured candidate YY ♀ physiology superfemale fish and the common milter mating of XY ♂ in the operation 3.3,, should produce 100% milter, the common milter of 50%XY ♂ wherein, 50%YY ♂ supermale fish because of chromosome (gene) reorganization.And 25%XX ♀ raun then appears in the common milter mating of XY ♀ physiology raun and XY ♂, 75% milter (wherein 50%XY ♂, 25%YY ♂).That is: YY ♀ * XY ♂ → 50%XY ♂+50%YY ♂, XY ♀ * XY ♂ → 25%XX ♀+50%XY ♂+25%YY ♂.
4.3 screening, identify: according to the analysis of two mating combination offspring test crosses, can identify the genetic sex of candidate tested fish, filter out YY ♂ supermale fish and YY ♀ physiology superfemale fish is standby.
5. expand the group: be the quick expansion group who utilizes YY ♂ supermale fish and YY ♀ physiology superfemale fish breeding population, get the YY ♂ supermale fish and the mating of YY ♀ physiology superfemale fish that have been identified in the operation 4, produce 100%YY ♂ supermale fish, when the fry opening, get a part of YY ♀ supermale fry by operation 2, become YY ♀ physiology superfemale fish with the reverse of oestrogenic hormone inductivity, promptly
The physiology superfemale fish.
6. the full milter production of hybrid seeds: with common raun of XX ♀ and the mating of YY ♂ supermale fish, the complete male yellow cartfish of the XY ♂ that then is produced on a large scale, i.e. XX ♀ * YY ♂ → full milter of 100%XY ♂.
Claims (1)
1. the method for supermale yellow cartfish breeding population is set up in supermale fish and the mating of physiology raun, comprise yellow cartfish artificial induced spawning, artificial insemination, male yellow cartfish sex reversal is become physiology raun, test cross, the feature of the method for this breeding population is to be undertaken by following operation:
(1) artificial induced spawning and artificial insemination: getting body surface does not have that wound, belly are expanded, soft common female yellow cartfish, uses ocyodinic HCG and LRH-A
2Every fish pectoral fin base portion is carried out a shot, and dosage is HCG800～1000IU+LRH-A
25～8 μ g/ tails are placed in 26 ℃ the water, can extrude ripe ovum after 24～28 hours, place on the ice bath low temperature to preserve standby it; Choose gonapophysis little red milter HCG injection and LRH-A
2The ocyodinic that reduces by half is dissected it and to be taken out spermary, with scissors it is shredded, and preserves the liquid filter wash with sperm again and goes out sperm, places on the ice bath low temperature to preserve standby it; Getting ovum mixes with sperm and carries out the dry method artificial insemination; Place the fertilization ovum 27 ℃ of biochemical incubator constant temperature to hatch into milter and female fry and whole membrane; After treating absorption of yolk at its, fry uses artemia larvae as opening erbium material;
(2) sex reversal: common male yellow cartfish sex reversal is become the physiology raun, with artemia larvae as the oestrogenic hormone carrier, it was soaked 1 hour in the 17-α of 100～200 μ g/L ethinyl estradiol solution, suction filtration takes out the estrogenic halogen worm of the enrichment artificial propagation yellow cartfish seedling of initial feeding of throwing something and feeding again, every morning, afternoon and evening respectively throw something and feed once, feeding volume is as the criterion to eat up in 30 minutes, threw something and fed continuously 40 days, about 50% XY ♂ milter sex reversal becomes XY ♀ physiology raun in the fry that survives after female sex hormone is handled, and XX ♀ raun is constant, the juvenile fish of sex reversal is transferred in the cement pit supports to sexual maturity with the soft-shelled turtle feed;
(3) test cross and sex reversal: with XY ♀ physiology raun and YY ♂ supermale fish or the common milter test cross of XY ♂, screening XY ♀ physiology raun
(3.1) artificial induced spawning: get sexually matured candidate XY ♀ physiology raun and common raun and carry out conventional artificial induced spawning;
(3.2) artificial insemination: the female fish egg of every tail is divided into two parts, a and XY ♂ milter sperm insemination, another part inseminated with YY ♂ supermale milt;
(3.3) property is than analyzing: candidate supermale fish and the standby and sex reversal of physiology superfemale fish---after the fertilization ovum hatches fry, the fry of each mating combination is divided into two parts again, support temporarily in net cage respectively, get wherein 1/3rd fries with behind artemia larvae and the soft-shelled turtle forage feed to three month, take out 30～50 tail juvenile fish by tail lumbar injection glacial acetic acid fixer, just can differentiate spermary or ovary from the sexual gland form after the dissection, analyze sex ratio; Remaining juvenile fish moves in the cement pit foster standby as the YY ♂ supermale fish of candidate to sexual maturity; 2/3rds fry in addition, the method for using the oestrogenic hormone inductivity to reverse moves into after oestrogenic hormone is handled 40 days again and supports sexual maturity in the cement pit, and is standby as the YY ♀ physiology superfemale fish of candidate;
(3.4) other affirmation of candidate piscinity
(3.4.1) test cross of XY ♀ physiology raun and the affirmation of property than analysis and XY ♀ physiology raun, with XY ♀ physiology raun and the common milter mating of XY ♂, produce the common raun of 25%XX ♀, 50%XY ♂ milter and 25%YY ♂ supermale fish, the property than analyzing the result, confirm XY ♀ physiology raun, i.e. XY ♀ * XY ♂ → 25%XX ♀+50%XY ♂+25%YY ♂;
(3.4.2) affirmation of offspring's test cross of the common raun of XX ♀, the analysis of property ratio and the common raun of XX ♀: with common raun of XX ♀ and the common milter mating of XY ♂, its offspring's male and female fish respectively is 50%, can confirm than analyzing whether tested fish is the common raun of XX ♀ according to property, be XX ♀ * XY ♂ → 50%XX ♀+50%XY ♂, the sex ratios that compare two mating combinations, just can determine the genetic sex of candidate fish, it is standby to filter out XY ♀ physiology raun, and what be confirmed to be the common raun of XX ♀ abandons it;
(4) test cross, screening, evaluation YY ♂ supermale fish and YY ♀ physiology superfemale fish
(4.1) mensuration of YY ♂ supermale fish is got operation (3.3) sexually matured YY ♂ supermale fish and the common raun mating of XX ♀, as XX ♀ * YY ♂ → 100%XY ♂, can confirm that then tested fish is a YY ♂ supermale fish, as XX ♀ * XY ♂ → 50%XX ♀+50%XY ♂, can confirm that then tested fish is the common milter of XY ♂;
(4.2) test cross of YY ♀ physiology superfemale fish is got the sexually matured candidate YY of operation (3.3) ♀ physiology superfemale fish and the common milter mating of XY ♂, and then YY ♀ * XY ♂ → 50%XY ♂+50%YY ♂ promptly produces 100% milter; And the common milter mating of XY ♀ physiology raun and XY ♂, then XY ♀ * XY ♂ → 25%XX ♀+50%XY ♂+25%YY ♂ promptly produces common raun of 25%XX ♀ and 75%XY ♂ milter;
(4.3) screening, identify: than analyzing, can identify the genetic sex of candidate tested fish according to two mating combination offspring test crosses, it is standby to filter out YY ♂ supermale fish and YY ♀ physiology superfemale fish;
(5) expand the group: the quick expansion group of YY ♂ supermale fish and YY ♀ physiology superfemale fish breeding population, get YY ♂ supermale fish and the mating of YY ♀ physiology superfemale fish that operation (4) has been identified, produce 100%YY ♂ supermale fish, when the fry opening, getting a part of YY ♂ supermale fry becomes YY ♀ physiology superfemale fish by operation (2) with the reverse of oestrogenic hormone inductivity, promptly
The physiology superfemale fish;
(6) the full milter production of hybrid seeds: with common raun of XX ♀ and the mating of YY ♂ supermale fish, the complete male yellow cartfish of the XY ♂ that then is produced on a large scale, i.e. XX ♀ * complete male yellow cartfish of YY ♂ → 100%XY ♂.
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