CN101002546A - Method for inducing sex reversal of Banhuashe soleidae fishes - Google Patents
Method for inducing sex reversal of Banhuashe soleidae fishes Download PDFInfo
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Abstract
A method for inducing the sex reversal of half-smooth tongue-sole including temp inducing and hormone inducing is disclosed. Said temp inducing method features that the fry is cultured at 28-32 deg.C for 50-60 days. Said hormone inducing method is characterized by that the fry is immersed in the aqueous solution of methyl testosterone for 50-60 days.
Description
Technical field
Present technique belongs to the fish genetic breeding technical field, is a kind of new method that the pseudo-milter of Cynoglossus semilaevis male on female, the physiology is gone up in heredity of cultivating, and can be applicable to fish sex control and complete female seed rearing.
Background technology
Cynoglossus semilaevis (Cynoglossus semiliaevis) is the distinctive a kind of famous and precious economic seawater fish of China, belongs to coastal waters warm water demersal fishes, and China is coastal all distribution, is many with the Huanghai Sea, the Bohai Sea.Cynoglossus semilaevis is because its delicious flavour, and fine and tender taste is nutritious, welcome by consumers in general, and its market value is high, and the breed prospect is boundless.In recent years, China scientific and technical personnel have carried out big quantity research to Cynoglossus semilaevis artificial propagation and genital regulating, and broke through Cynoglossus semilaevis artificial breeding technique (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science first in 1993, Jiang Yanwei etc., 1993), having carried out Cynoglossus semilaevis subsequently propagates artificially and the genital regulating Study on Technology, set up the Cynoglossus semilaevis parent fish rearing, adopt ovum technology and semi-smooth tongue sole offspring breed production technology (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, Liu Xuezhou etc., 2006), also carried out Cynoglossus semilaevis chromosome analysis (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science simultaneously, Zhou Liqing etc., 2005) and a research work such as Isozyme Analysis (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, village will is violent etc., 2006).But studies show that there are huge difference in Cynoglossus semilaevis female individuals and male on growth rate, its female individuals is than the big 2-3 of male times (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, Meng Tianxiang etc., 1988).Because it is slow that male was grown, reduced the cultured output of Cynoglossus semilaevis, increase aquaculture cost, thereby had a strong impact on the popularization of semi-smooth tongue sole offspring breed and the formation of aquaculture industry.So far, about Cynoglossus semilaevis sex reversal and sex controlling Study on Technology, there is no report both at home and abroad.
Summary of the invention:
The objective of the invention is by the influence of the research environment factor (as temperature etc.) and exogenous hormones Cynoglossus semilaevis gonad development and Sex Differentiation, understand fully the temperature condition and hormone kind and the concentration that influence the Cynoglossus semilaevis sex reversal, the technical method of Cynoglossus semilaevis sex reversal is induced in foundation, for Cynoglossus semilaevis sex controlling and complete female breeding research provide technological means.
Its technology contents of the present invention is as follows:
Comprise two aspects: one, the method for temperature-induced Cynoglossus semilaevis sex reversal; Two, exogenous hormone is induced the method for Cynoglossus semilaevis sex reversal.
One, the method for temperature-induced Cynoglossus semilaevis sex reversal: its technology contents comprises: 1, the fry developmental stage of suitability reverse determines; 2, induce the screening of the preference temperature of Cynoglossus semilaevis sex reversal; 3, the analysis of Cynoglossus semilaevis sex reversal individuality:
1, determining of the fry developmental stage of suitability reverse:
Cut into slices by paraffin organization, double early stage sexual gland atomization of sliding-tongue sole seedling carries out histological observation and finds: under 24 ℃ of raising conditions, the Cynoglossus semilaevis fry begins differentiation at back 30 days left and right sides sexual glands of hatching, and the raun sexual gland is to the differentiation of ovary direction, and the milter sexual gland breaks up to the spermary direction.Therefore, we have determined to induce effective zero-time of Cynoglossus semilaevis sex reversal should be for hatching the back 20-30 days.
2, induce the preference temperature and the screening of processing duration of Cynoglossus semilaevis sex reversal:
25-100 days double sliding-tongue sole seedling carries out different temperatures (16 ℃, 20 ℃, 24 ℃, 28 ℃, 32 ℃) processing after hatching, continue to handle 50-60 days, the sexual gland of fry after the acquisition process, utilize the paraffin organization section to identify phenotypic sex, observe the influence of temperature to the sexual gland differentiation, the result shows: 28 ℃ and 32 ℃ of high temperature can be induced the transformation of Cynoglossus semilaevis female individuals to male, improve the male ratio in the colony, reach 69.2% and 66.7% respectively; And 16 ℃ of low temperature and 20 ℃ are little to the Sex Differentiation influence, and male ratio is respectively 56.5%, 57.1% in the colony; The individual ratio of male and female was near 1: 1 in 24 ℃ of processing colonies.Thereby we find, and to induce Cynoglossus semilaevis fry sexual gland be 28 ℃-32 ℃ by female effective temperature to male conversion.
3, the analysis of Cynoglossus semilaevis sex reversal individuality:
Adopt paraffin organization sections observation gonadal cell microstructure to identify phenotypic sex, the Cynoglossus semilaevis liver dna that extracts the process Temperature Treatment simultaneously is as template, adopt round pcr, utilize a pair of Cynoglossus semilaevis female individuals specific PCR primer, (primer 1:5 '-ATTCACTGACCCCTGAGAGC-3 '; Primer 2: 5 '-AACAACTCACACACGAC AAATG-3 '), carry out pcr amplification, identify the genetic sex of Cynoglossus semilaevis.To have genetic sex be female and phenotype is male individuality is that sex is male sex reversal individuality by female sex reversal.
Two, exogenous hormone is induced the method for Cynoglossus semilaevis sex reversal: its technology contents comprises: 1, the fry developmental stage of suitable HORMONE TREATMENT inductivity reverse determines; 2, induce the screening of the sex hormone concentration of Cynoglossus semilaevis sex reversal; 3, the Cynoglossus semilaevis hormonal reverses individual analysis:
1, determining of the fry developmental stage of suitable HORMONE TREATMENT inductivity reverse:
Cut into slices by paraffin organization, the early stage sexual gland differentiation carrying out of Cynoglossus semilaevis histological observation is found: under 24 ℃ of raising conditions, hatch back 30 days Cynoglossus semilaevis sexual glands and begin differentiation, therefore we determine to induce the HORMONE TREATMENT time of Cynoglossus semilaevis sex reversal should be for hatching in back 30 days, because inducing of fish sex reversal must be carried out before the sexual gland differentiation.
2, induce the sex hormone concentration of Cynoglossus semilaevis sex reversal and the screening of processing mode:
25-100 days double sliding-tongue sole seedling carries out variable concentrations gradient (20 μ g/LH after hatching
2O, 30 μ g/LH
2O, 50 μ g/LH
2O, 80 μ g/LH
2O, 100 μ g/L.H
2O) androgen methyltestosterone dipping bath is handled, after hatching, utilize during 9 monthly ages the paraffin organization section to identify phenotypic sex, observe the influence of hormone to the sexual gland differentiation, the result shows: can significantly improve male ratio in the colony in all HORMONE TREATMENT groups, reach 100%, 97.7%, 100%, 97.4%, 100% respectively.Thereby effectively to induce Cynoglossus semilaevis fry sexual gland be 20-100 μ g/LH by female sex hormone concentration and processing mode to male conversion
2The methyltestosterone dipping bath of O concentration is handled the Cynoglossus semilaevis fry.
3, the Cynoglossus semilaevis hormonal reverses individual analysis:
Adopt paraffin organization sections observation gonadal cell microstructure to identify its physiology sex, extract through hormone-treated Cynoglossus semilaevis liver dna simultaneously as template, adopt round pcr, utilize a pair of Cynoglossus semilaevis female individuals specific PCR primer, (primer 1:5 '-ATTCACTGACCCCTGAGAGC-3 '; Primer 2: 5 '-AACAACTCACACA CGAC AAATG-3 '), carry out pcr amplification, identify the genetic sex of Cynoglossus semilaevis.To have genetic sex be female and phenotype is male individuality is that sex is male sex reversal individuality by female sex reversal.
The present invention and prior art contrast are characterized in: the present invention screens preference temperature, the suitability hormone concentration that the female individuals of inducing in the Cynoglossus semilaevis heredity is converted into physiological male, has set up first and has induced Cynoglossus semilaevis by the technical method of female individuals to the male sex reversal.This technology has simply, easy advantage such as capable, efficient, reliable, for new technological approaches has been opened up in fish sex control and unisexuality breeding, suit in Cynoglossus semilaevis and other cultured fishes, to apply, to cultured fishes sex controlling and the significant and using value of unisexuality breeding.
Description of drawings:
Fig. 1, temperature are to the influence of Cynoglossus semilaevis Sex Differentiation
* difference not remarkable (P＞0.05) is compared in expression with control group (24 ℃); * represents that significant difference is arranged compared with the control (0.01＜P＜0.05, Chi-square Test)
Fig. 2: the gonadal tissue section of temperature-induced Cynoglossus semilaevis sex reversal individuality;
A:32 ℃ of processing; B:28 ℃ of processing
Fig. 3: the genetic sex analysis of temperature-induced Cynoglossus semilaevis sex reversal individuality;
F: normal female; MS: sex reversal is male; M: normal male
Fig. 4: androgen is induced the gonadal tissue section of Cynoglossus semilaevis sex reversal individuality;
C： 30μg/L·H
2O； D：80μg/L·H
2O
Fig. 5: androgen is induced the genetic sex analysis of Cynoglossus semilaevis sex reversal individuality;
M:Marker; 1: female contrast; 2:MT handles female; 3: male contrast; 4:MT handles male; The sex reversal individuality that 5-8:MT induces.
Embodiment:
Adopt ecology regulation and control and technological guide technology, set up the technical method that Cynoglossus semilaevis inheritance reverses, this is for Cynoglossus semilaevis sex controlling, the complete female seed of cultivation Cynoglossus semilaevis, carrying out complete female seed cultures, improve the cultured output of Cynoglossus semilaevis, improve the economic benefit of culturing, really Cynoglossus semilaevis is developed as a good breed variety of being accepted by numerous raisers, promote the development of cynoglossus semilaevis cultivation industry and the update of marine fish culture kind, have important practical significance and huge economic benefit.
Embodiment below by ' abductive approach of Cynoglossus semilaevis sex reversal ' is elaborated to technology contents of the present invention in conjunction with the accompanying drawings:
Its technology contents of the present invention comprises two aspects: one, the method for temperature-induced Cynoglossus semilaevis sex reversal; Two, exogenous hormone is induced the method for Cynoglossus semilaevis sex reversal.
One, the method for temperature-induced Cynoglossus semilaevis sex reversal: its technology contents comprises: 1, the fry developmental stage of suitability reverse determines; 2, induce the screening of the preference temperature of Cynoglossus semilaevis sex reversal; 3, the analysis of Cynoglossus semilaevis sex reversal individuality:
1, determining of the fry developmental stage of suitability reverse:
The Cynoglossus semilaevis parent population through support temporarily, domestication, artificial induction's gonad maturity, female, male parent population ovulates, arrange the fry that essence obtains natural spawning fertilization simultaneously.Under 24 ℃ of raising conditions, 10,20,30,45,50,75 days 5-10 bars of taking a sample respectively after hatching, Bouin ' s is liquid-solid fixed.Cut into slices by paraffin organization, to the early stage sexual gland differentiation carrying out of Cynoglossus semilaevis histological observation, Cynoglossus semilaevis is in back 10 days of hatching and 20 days all visible Gonadal rudiment of microscopically, and original hase is positioned at the kidney duct below, the enteron aisle top, original hase does not all discover a marked discrepancy on cytology and morphology.Differentiation occurred at back 30 days sexual glands of hatching, two kinds of tangible sexual gland types have appearred in gonadal tissue, do not have the crack in a kind of Gonadal rudiment; The crack appears in another kind of original hase central authorities, and following growth of Gonadal rudiment that wherein has the crack is ovary, is not spermary and do not have following a growth of Gonadal rudiment in crack.Thereby think that the histology sexual gland differentiation of Cynoglossus semilaevis began in hatching in back 30 days, the temperature that studies show that of multiple fish must be carried out before the differentiation of histology sexual gland the sexual gland differentiation, and must overlap at least this critical period, thereby induce the time of Cynoglossus semilaevis sex reversal should beginning in back 30 days of hatching, be preferably in hatching and begin to carry out Temperature Treatment during back 20 days-30 days.
2, induce the screening of the preference temperature of Cynoglossus semilaevis sex reversal:
After hatching 25-100 days, adopt 16 ℃, 20 ℃, 24 ℃, 28 ℃, 32 ℃ water temperatures to raise the Cynoglossus semilaevis prelarvas respectively.In hatching back September to each group of each temperature groups choose 5-10 bar fish get its Liver Allograft Preservation in-80 ℃ standby, and it is liquid-solid fixed with Bouin ' s to get sexual gland.The prelarva and the sexual gland that take out are made paraffin section, and through ethanol dehydrations at different levels, dimethylbenzene is transparent and paraffin embedding, does continuous crosscut section, slice thickness is 5～6 μ m.H.E dyeing, the Olympus observation by light microscope is differentiated its phenotypic sex.When 9 monthly ages, Cynoglossus semilaevis spermary and ovary profile and exterior color significant difference, ovary is elongated, and appearance is a milky; And the spermary front end is thicker, sharply diminishes backward, forms conical in shape, and color is darker, and appearance is a light/dark balance.According to this significantly different male and female proterties, adopt high light transmission live body Cynoglossus semilaevis sexual gland position, can distinguish the phenotypic sex of Cynoglossus semilaevis live body.50 tail fishes of picking are observed male and female result that the sexual gland microstructure identifies with paraffin section at random also proves and takes high light transmission sexual gland position, judges that according to its sexual gland color and proterties the phenotypic sex of this fish is feasible.28 ℃ and 32 ℃ of high temperature can significantly improve the male ratio in the colony, reach 69.2%, 66.7% respectively; And 16 ℃ of low temperature and 20 ℃ are little to the Sex Differentiation influence, and male ratio is respectively 56.5%, 57.1% in the colony; The individual ratio of male and female was near 1: 1 in 24 ℃ of processing colonies.Thereby adopting 28 ℃ and 32 ℃, can effectively to induce Cynoglossus semilaevis be the male (see figure 1) of phenotype by the female sex reversal in the heredity.
Temperature is to the influence of Cynoglossus semilaevis Sex Differentiation, under 16 ℃, 20 ℃, 24 ℃, 28 ℃, 32 ℃ (N=23,42,81,78,93) conditions, male ratio is respectively 56.5%, 57.1%, 50.6%, 69.2%, 66.7%, and the female-male proportion of control group (24 ℃) was near 1: 1
3, the analysis of Cynoglossus semilaevis sex reversal individuality:
7-9 is during the monthly age after hatching, adopt paraffin organization sections observation gonadal cell microstructure to identify the phenotypic sex of Cynoglossus semilaevis, the Cynoglossus semilaevis liver dna that extracts the process Temperature Treatment simultaneously is as template, adopt round pcr, utilize a pair of Cynoglossus semilaevis female individuals specific PCR primer (primer 1:5 '-ATTCACTGACCCCTGAGAGC-3 '; Primer 2: 5 '-AACAACTCACACACGACAAATG-3 '), carry out pcr amplification, identify the genetic sex of Cynoglossus semilaevis, can amplify the female individuals in the heredity that is of specific DNA fragment, can not amplify the male in the heredity that is of specific DNA fragment; To have genetic sex be female and phenotype is male individuality is that sex is male sex reversal individuality by female sex reversal.We have found the pseudo-milter individuality of sex reversal, and the qualification result of its physiology sex and genetic sex is seen Fig. 2 and Fig. 3.
Two, exogenous hormone is induced the method for Cynoglossus semilaevis sex reversal: its technology contents comprises: 1) the fry developmental stage of suitable HORMONE TREATMENT inductivity reverse determines; 2) induce the screening of the sex hormone concentration of Cynoglossus semilaevis sex reversal; 3) the Cynoglossus semilaevis hormonal reverses individual analysis:
1) determining of the fry developmental stage of suitable HORMONE TREATMENT inductivity reverse:
Identical with the developmental stage of temperature-induced sex reversal;
2) induce the sex hormone concentration of Cynoglossus semilaevis sex reversal and the screening of processing mode:
After hatching, Cynoglossus semilaevis carried out variable concentrations gradient (20 μ g/LH in 25-100 days
2O, 30 μ g/LH
2O, 50 μ g/LH
2O, 80 μ g/LH
2O, 10 μ g/LH
2O) androgen methyltestosterone dipping bath is handled, in hatching back September to each group of each temperature groups choose 5-10 bar fish get its Liver Allograft Preservation in-80 ℃ standby, and it is liquid-solid fixed to get sexual gland Bouin ' s.The prelarva and the sexual gland that take out are made paraffin section, and through ethanol dehydrations at different levels, dimethylbenzene is transparent and paraffin embedding, does continuous crosscut section, slice thickness is 5～6 μ m.H.E dyeing, the Olympus observation by light microscope is differentiated its phenotypic sex.When 9 monthly ages, Cynoglossus semilaevis spermary and ovary profile and exterior color significant difference, ovary is elongated, and appearance is a milky; And the spermary front end is thicker, sharply diminishes backward, forms conical in shape, and color is darker, and appearance is a light/dark balance.According to this significantly different male and female proterties, adopt high light transmission live body Cynoglossus semilaevis sexual gland position, can distinguish the phenotypic sex of Cynoglossus semilaevis live body.50 tail fishes of picking are observed male and female result that the sexual gland microstructure identifies with paraffin section at random also proves and takes high light transmission sexual gland position, judges that according to its sexual gland color and proterties the phenotypic sex of this fish is feasible.The result shows: at all HORMONE TREATMENT groups (20 μ g/LH
2O, 30 μ g/LH
2O, 50 μ g/LH
2O, 80 μ g/LH
2O, 100 μ g/LH
2O) can both significantly improve male ratio in the colony, reach 100%, 97.7%, 100%, 97.4%, 100% respectively.Thereby employing 20-100 μ g/LH
2The methyltestosterone dipping bath of O concentration is handled the Cynoglossus semilaevis fry, can effectively induce Cynoglossus semilaevis to take place by female to male sex conversion.(seeing Table 1)
Table 1: variable concentrations methyltestosterone dipping bath is handled the influence to the Cynoglossus semilaevis Sex Differentiation
| Androgen concentration (μ g/LH 2O) | Sum (mantissa) | Male (mantissa) | Female (mantissa) | Male ratio (%) |
| 20 30 50 80 100 | 58 43 12 38 39 | 58 42 12 38 39 | 0 1 0 1 0 | 100 97.7 100 97.4 100 |
3) the Cynoglossus semilaevis hormonal reverses individual analysis:
7-9 is during the monthly age after hatching, adopt paraffin organization sections observation gonadal cell microstructure to identify the phenotypic sex of Cynoglossus semilaevis, extract through hormone-treated Cynoglossus semilaevis fry liver dna simultaneously as template, adopt round pcr, utilize a pair of Cynoglossus semilaevis female individuals specific PCR primer (primer 1:5 '-ATTCACTGACCCCTGAGAGC-3 '; Primer 2: 5 '-AACAACTCACACACGAC AAATG-3 '), carry out pcr amplification, identify the genetic sex of Cynoglossus semilaevis, can amplify the female individuals in the heredity that is of specific DNA fragment, can not amplify the male in the heredity that is of specific DNA fragment; To have genetic sex be female and phenotype is male individuality is that sex is male sex reversal individuality (seeing Fig. 4 and Fig. 5) by female sex reversal.
Claims (2)
1. the method for a temperature-induced Cynoglossus semilaevis sex reversal is characterized in that its method of operating comprises: the screening of the preference temperature of determining, inducing the Cynoglossus semilaevis sex reversal of the fry developmental stage that suitability reverses, the analysis of Cynoglossus semilaevis sex reversal individuality: described fry developmental stage of determining to induce the sex reversal of Cynoglossus semilaevis fry: for hatching back 20 days-30 days; Described screening induces the Cynoglossus semilaevis fry that the effective temperature of sex reversal takes place: be 28-32 ℃, handling the duration is 50-60 days; The analysis of described Cynoglossus semilaevis sex reversal individuality: adopt paraffin section method to identify the physiology sex of sex reversal fish; Adopt the specific PCR method to identify the genetic sex of sex reversal fish, promptly utilize a pair of Cynoglossus semilaevis female individuals specific PCR primer, carry out pcr amplification, primer 1:5 '-ATTCACTGACCCCTGAGAGC-3 '; Primer 2: 5 '-AACAACTCACACACGAC AAATG-3 '.
2. the method for hormone induction Cynoglossus semilaevis sex reversal, the method of operating that it is characterized in that it comprises: the screening of the sex hormone concentration of determining, inducing the Cynoglossus semilaevis sex reversal of the fry developmental stage that suitable HORMONE TREATMENT inductivity reverses, the individual analysis of Cynoglossus semilaevis hormonal reverse, and described definite suitable fry developmental stage of hormone induction Cynoglossus semilaevis fry sex reversal that carries out is for hatching the back 20-30 days; Effective hormone kind and concentration that described screening induces the Cynoglossus semilaevis fry that sex reversal takes place are methyltestosterone, and hormone concentration is 20-100 μ g/LH
2O, the hormone occupation mode is for soaking, and soak time is 50-60 days; It is to adopt paraffin section method to identify the physiology sex of sex reversal fish that described Cynoglossus semilaevis hormonal reverses individual analysis, adopt the specific PCR method to identify the genetic sex of sex reversal fish: promptly to utilize a pair of Cynoglossus semilaevis female individuals specific PCR primer, carry out pcr amplification, primer 1:5 '-ATTCACTGACCCCTGAGAGC-3 '; Primer 2: 5 '-AACAACTCACACA CGACAAATG-3 '.
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