CN103529036A - Sex identification method for juvenile fish of hybrid pelteobagrus fulvidraco (richardson) - Google Patents
Sex identification method for juvenile fish of hybrid pelteobagrus fulvidraco (richardson) Download PDFInfo
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- CN103529036A CN103529036A CN201310466149.4A CN201310466149A CN103529036A CN 103529036 A CN103529036 A CN 103529036A CN 201310466149 A CN201310466149 A CN 201310466149A CN 103529036 A CN103529036 A CN 103529036A
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Abstract
The invention discloses a sex identification method for juvenile fish of hybrid pelteobagrus fulvidraco (richardson) [pelteobagrus fulvidraco (richardson) (male)* pelteobagrus vachelli (richardson) (female)]. The hybrid pelteobagrus fulvidraco (richardson) having an individual body length of above 3 cm is identified by utilization of a method of living-body gonad squashing, acetocarmine staining and ethanol crystal violet staining, and the accuracy of the method is verified by a paraffin section of the gonad tissue. The sex identification method is suitable for sex identification for juvenile fish of common pelteobagrus fulvidraco (richardson) and of "all-male No.1" pelteobagrus fulvidraco (richardson). By the sex identification method, sex identification of one juvenile fish can be completed in 3 min, and 100 fishes can be identified by cooperation of two persons for 2 h. The sex identification method has characteristics of high efficiency, economy, rapidness, accuracy and stability. The sex identification method has important application value for male fry cultivation of the pelteobagrus fulvidraco (richardson) and the hybrid pelteobagrus fulvidraco (richardson) thereof, and can be applied for rapid sex identification of a pelteobagrus fulvidraco (richardson) commodity summer fry.
Description
Technical field
The invention belongs to the fish sex authenticate technology in aquatic biological technical field, in particular, the present invention relates to a kind of hybridization Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)] juvenile fish property method for distinguishing of quick and precisely differentiating, the method is equally applicable to the sex identification of common Pelteobagrus fulvidraco and " complete male No. one " parr of yellow catfish.The present invention, have important using value in Pelteobagrus fulvidraco and the male seedling fostering of hybridization Pelteobagrus fulvidraco thereof, can be applied to the Rapid identification of Pelteobagrus fulvidraco commodity seedling sex.
Background technology
At present, Pelteobagrus fulvidraco breed variety mainly contains two kinds, hybridizes Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)], " complete male No. 1 " Pelteobagrus fulvidraco.
Pelteobagrus fulvidraco [Pelteobagrus fulvidraco(Richardson)] and Pelteobagrus vachelli [Pelteobagrus vachelli(Richardson)] be all under the jurisdiction of SILURIFORMES (Siluriformes) ,Chang section (Bagridae), Pelteobagrus (Pelteobagrus) fish, be two higher kinds of economic worth in 5 kinds that Pelteobagrus is known.In recent years, along with the breakthrough of Pelteobagrus fulvidraco and Pelteobagrus vachelli artificial propagation and cultural technique, propagating artificially on a large scale just throughout the country of these two kinds of fishes risen, particularly Pelteobagrus fulvidraco propagate artificially more extensive.Yet, these two kinds of fishes exist larger difference on biological and ecological characteristics: Pelteobagrus fulvidraco is mainly distributed in lake, hypoxia-resistant capacity is higher than Pelteobagrus vachelli, be suitable for pond culture. but its individuality is less, the speed of growth is slower, in highdensity pond culture, then seed more difficult grow to listing specification (more than 50g); Pelteobagrus vachelli is mainly distributed in the flowing water water body of rivers, the a kind of of individual maximum in Pelteobagrus, 2 age fish body weight can reach 150-600g, maximum whose body weight reaches 1850g, its fast growth, growing sturdily then kind can grow to listing specification, but that its shortcoming is hypoxia-resistant capacity is poor compared with Pelteobagrus fulvidraco, in pond culture, when water temperature surpasses 32 ℃, mortality ratio rises greatly.Although Culture of Pelteobagrus Fulvidraco has been the new growth engines of many local fishery's production developments at present, numerous fishermen still urgently wish the appearance of Pelteobagrus fulvidraco new varieties.The utilization of heterosis, hybrid vigor has become the important channel of improving fish crop and improving fish products matter, the hybrid generation obtaining by Pelteobagrus fulvidraco and Pelteobagrus vachelli hybridization, have body colour tempting (tawny), growth fast, strong stress resistance (as than Pelteobagrus vachelli lower oxygen concentration resistance and high temperature resistant), seed can be formed the advantages such as listing specification then, has very much development potentiality.
Under identical cultivating condition, the male Pelteobagrus fulvidraco of First Year is than the fast growth of raun born of the same parents 30% left and right, and at the Second Year of cultivation, its milter grows to 150-200g, and raun only has 50-70g, and male and female fish growth differences approaches 3 times.Therefore, if can be in breeding the sex of manual control Pelteobagrus fulvidraco, cultivate complete male Pelteobagrus fulvidraco, will greatly improve output, reduce the cost of breeding fish, increase economic efficiency.Inst. of Hydrobiology, Chinese Academy of Sciences's usability reverses the YY supermale Pelteobagrus fulvidraco that obtains can be used for production with gynogenesis, carries out artificial propagation with common Pelteobagrus fulvidraco raun (XX), has obtained complete male Pelteobagrus fulvidraco (XY).
The early sexes of fish is identified and controlled is the significant problem that fishery study field is paid close attention to always, and sex identification is accompanied by the in-depth to Sex Determination molecular mechanism research, research method by classic method to molecular biology method transition.At present, in most fish, can not identify atypia chromosome, so many scholars directly observe sex chromosome from cellular level, but the method often exists artificial subjective factor easily to lead to errors, mainly by chromosome banding, sex-linked inheritance proterties and induction thelykaryon or androgenesis etc., carry out distinctiveness chromosome now; Some endangered species are as inadvisable in the direct anatomic observation sexual gland of Acipenseridae (Acipenseridae) fish, and conventional method is Minimally Invasive Surgery method, endoscope, ultrasonic identification method, blood biochemical and Hormone traits diagnostic method and identified for genes method etc.These authentication method complex steps, and need operator to possess darker Protocols in Molecular Biology and some distinctive instruments could obtain result.
At present, the sex appraisal method of hybridization Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)], common Pelteobagrus fulvidraco, " complete male No. one " Pelteobagrus fulvidraco is many to be identified from morphology aspect, yet morphological method can not effectively be identified at young stage, can only just can carry out in the adult fish phase, but it is very important that the feed that Pelteobagrus fulvidraco male and female fish growth differences causes expends for the department of growing seedlings and raiser, in the urgent need at seed young stage just by sex identification out.Along with developing rapidly of modern molecular biology, with SRAP, AFLP equimolecular mark, parr of yellow catfish sex is identified, but authentication method complex steps, and need operator to possess the instruments such as professional Protocols in Molecular Biology and PCR instrument, electrophoresis apparatus, gel imaging instrument could to obtain desirable result.
In sum, need at present a kind of economy, fast, Pelteobagrus fulvidraco and cenospecies male and female discrimination method thereof are applied to production practices accurately, the present invention only need to grow to more than 3 centimetres juvenile fish, from juvenile fish colony, sampling is just easy to distinguish juvenile fish sex at random, and the sex identification that the method completes 1 tail juvenile fish only needs 3 minutes, 2 people coordinate 2 hours and can detect 100 tail juvenile fish, and rate of accuracy reached to 100%, greatly save man power and material, can be promoted and apply in Pelteobagrus fulvidraco sex identification field.
Summary of the invention
The object of this invention is to provide a kind of hybridization Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)] juvenile fish property method for distinguishing of quick and precisely differentiating, and be applicable to the sex identification of common Pelteobagrus fulvidraco and " complete male No. one " parr of yellow catfish, be applicable to production practices, to overcome the defects such as the existing technology instrument demand of utilizing modern molecular biology technique to identify parr of yellow catfish sex is too high, detection cost is high, method step is loaded down with trivial details, authentication method of the present invention is more efficient, economical, quick, accurate, stable, is applicable to production practices.
For realizing object of the present invention, technical scheme of the present invention is:
Hybridization Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)] juvenile fish property method for distinguishing, the method comprising the steps of:
1) dyeing liquor glacial acetic acid fuchsin solution preparation step: take 0.5g fuchsin coloring agent, add the glacial acetic acid of 100mL45%, heating for dissolving 2-4 minute; After cooling, cross leaching filtrate, lucifuge normal temperature is preserved;
2) the purple solution preparation step of dyeing liquor alcohol crystal: take violet staining agent 1.0g, ammonium oxalate 0.4g, and then add the ethanol of 20mL95%, and stirring and dissolving, finally adds 80mL distilled water to mix, and gets its filtrate after filtration, and lucifuge normal temperature is preserved;
3) gonadal tissue fixing step: with 5% acetum 0.2-0.4mL stationarity glandular tissue, gonadal tissue is fixed rapidly, quality hardening, color becomes milky;
4) gonadal tissue sampling procedure: after fixing, 30-60 takes out milky gonadal tissue in second, be placed on and drip in advance on the microslide that has 1 fuchsin acetum, add again 1 crystal violet ethanolic solution by its dyeing 2-4 minute, last covered also flattens gonadal tissue gently, is placed on 4 * 10 biology microscope Microscopic observation;
5) compressing tablet is observed: the sex of differentiating juvenile fish according to the difference of the aspects such as juvenile fish sexual gland shape, cell arrangement mode, cell individual size; Sexual gland shape is comb shape branch; Cell volume is little, and cell is together tightly packed, and cell and cell compartment are very indistinguishable is milter; Sexual gland shape is fiber ligature; Cell volume is relatively large, the rounded or oval of shape, cell arrangement rule, clear-cut be raun.
The invention also discloses in the method ，Shi juvenile fish colony that utilizes said method to measure hybridization Pelteobagrus fulvidraco male ratio and carry out random sampling, sample size is more than 100 tails, and the individual body long size of juvenile fish being sampled is more than 3 centimetres; Individuality to sampling is differentiated sex according to the method described above; According to identification result, draw the male ratio of juvenile fish colony.
In order to verify the accuracy of the inventive method, by same individual another gonadal tissue take out and adopt traditional BouinShi immobile liquid according to the fixing juvenile fish sexual gland of conventional method, then carry out routine paraffin wax section, after rear utilization hematoxylin eosin staining method dyeing, observe, with the comparison of the observable sex result of compressing tablet of the present invention.By comparing, two gonadal tissues that same tail juvenile fish is taken out are used respectively dyeing pressed-disc technique consistent with the resulting sex identification result of paraffin section technology, show the dye juvenile fish sex identification rate of accuracy reached to 100% of pressed-disc technique application hybridization Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)], common Pelteobagrus fulvidraco, " No. one, full hero " Pelteobagrus fulvidraco of the present invention.
Method of the present invention, hybridization Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)], common Pelteobagrus fulvidraco, " complete male No. one " parr of yellow catfish grow to 3 centimetres above can be efficient, economical, quick, accurate, stable the sexes of evaluation juvenile fish, because male and female fish growth differences approaches 3 times, the mensuration Pelteobagrus fulvidraco male ratio that production division and raiser can fast and stable that makes to grow seedlings, to increase the benefit of marketable fish.
Accompanying drawing explanation
Fig. 1, for hybridization Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)] spermary compressing tablet, shows spermatogonium (╳ 40).
Fig. 2, for hybridization Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)] ovary compressing tablet, shows oogonium (╳ 40).
Embodiment
Instantiation of the present invention is as follows:
1, identify early-stage preparations
1) medicine: 5% acetum, acetic acid (glacial acetic acid), fuchsin coloring agent (phenol red), violet staining agent, ammonium oxalate, 95% ethanol, distilled water
2) equipment: biological microscope, 1mL syringe, eye scissors, iris pincet, microslide, cover glass, dropper
3) dyeing liquor preparation: method preparation glacial acetic acid fuchsin solution, the purple solution of alcohol crystal introduced in by specification
2, sex identification:
Jiangsu Province Nanjing Normal University school of life and health sciences aquatic economic animal germ plasm resource and genetic breeding research chamber go out to hybridize Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)] 100,000 tails, common Pelteobagrus fulvidraco 20,000 tails, " complete male No. one " Pelteobagrus fulvidraco 50,000 tails in June, 2013 at Lu mouth Pelteobagrus fulvidraco Experimental Base artificial breeding.Hybridization Pelteobagrus fulvidraco is after opening after 30 days, and 90% fry grows to more than 3 centimetres; Pelteobagrus fulvidraco is after opening after 35 days for common Pelteobagrus fulvidraco and " complete male No. one ", and 90% fry grows to more than 3 centimetres, and each gets 100 tails at random 3 kinds of juvenile fish.
With syringe, 5% acetum is injected to the abdominal cavity of juvenile fish, employing pectoral fin injects or directly from Intraperitoneal injection, injection volume is 0.2-0.4mL, and the individual injection volume bigger than normal of juvenile fish will increase, and the gonadal tissue of fry is fixed rapidly, quality hardening, and color becomes milky.To gonadal tissue fixedly time, the injection volume of GPRS 5% acetum and sexual gland regular time.If the set time surpasses 1 minute, can cause the color of juvenile fish body cavity inner tissue to shoal, be difficult to gonadal tissue to distinguish, increase the difficulty of gonadal tissue sampling.When carrying out gonadal tissue sampling, once gonadal tissue is taken out with iris pincet, and not want other too much tissue of adhesion, otherwise the gonadal tissue of fry will be difficult to differentiate and take out once again.
After injection, in 30 seconds, with operating scissors, from anus, toward cephalad direction, cut off, cut to skull place, then use scissors with perpendicular to opening direction, the side of fish is cut off, as opening the door, with tweezers, push the muscle on side aside, push gently visceral mass aside, two gonadal tissues that bleach of being close to back peritonaeum are cut off to taking-up or directly pullled sexual gland one side by the disposable taking-up of sexual gland, be placed on and drip in advance on the microslide that has 1 (0.05mL) fuchsin acetum, add again 1 (0.05mL) crystal violet ethanolic solution by its dyeing 2-4 minute, last covered also flattens gonadal tissue gently, be placed on the biology microscope Microscopic observation of 4*10.Avoid dyeing time long (should be no more than 5 minutes), otherwise can cause gonadal tissue hyperchromatosis and increase observation difficulty, affect the accuracy that sex is differentiated.The solution concentration of fuchsin coloring agent is 0.5g/mL.The solution concentration of violet staining agent is 1g/mL.
According to the difference of the aspects such as juvenile fish sexual gland shape, cell arrangement mode, cell individual size, differentiate the sex of juvenile fish.By biology microscope Microscopic observation: milter spermary is organized painted dark compared with ovary tissue, and shape is comb shape branch (likeness in form dendroid); Spermatogonium volume is little, and cell is together tightly packed, and cell and cell compartment are difficult to differentiate (as shown in Figure 1).Raun ovary tissue is painted more shallow, and shape is fiber ligature; Oogonium volume is relatively large, the rounded or oval of shape, oogonium queueing discipline, clear-cut (as shown in Figure 2).
Adopt the accuracy of this invention of paraffin section technical identification: there is respectively a gonadal tissue every tail parr of yellow catfish body wall both sides, another gonadal tissue is taken out, with Bo Enshi immobile liquid according to the fixing juvenile fish sexual gland of conventional method, when getting juvenile fish sexual gland, cause use 5% acetic acid to fix, in order to guarantee composition and the concentration of immobile liquid, in fixation procedure, change immobile liquid 2 times.Then carry out routine paraffin wax section, after rear utilization hematoxylin eosin staining method dyeing, observe, with the observable juvenile fish sex of compressing tablet result comparison (in Table 1).
Table 1 dyeing pressed-disc technique and the comparison of paraffin section technical appraisement juvenile fish sex result
By relatively, with two gonadal tissues of tail juvenile fish taking-up, use respectively dyeing pressed-disc technique consistent with the resulting sex identification result of paraffin section technology.The sex identification rate of accuracy reached to 100% of this patented technology application hybridization Pelteobagrus fulvidraco [Pelteobagrus fulvidraco (♀) ╳ Pelteobagrus vachelli (♂)], common Pelteobagrus fulvidraco, " complete male No. one " Pelteobagrus fulvidraco.
Claims (2)
1. differentiate a hybridization parr of yellow catfish method for distinguishing, it comprises the following steps:
1) dyeing liquor glacial acetic acid fuchsin solution preparation step: take 0.5g fuchsin coloring agent, add the glacial acetic acid of 100mL45%, heating for dissolving 2-4 minute; After cooling, cross leaching filtrate, lucifuge normal temperature is preserved; 2) the purple solution preparation step of dyeing liquor alcohol crystal: take violet staining agent 1.0g, ammonium oxalate 0.4g, and then add the ethanol of 20mL95%, and stirring and dissolving, finally adds 80mL distilled water to mix, and gets its filtrate after filtration, and lucifuge normal temperature is preserved;
2) gonadal tissue fixing step: with 5% acetum 0.2-0.4mL stationarity glandular tissue, gonadal tissue is fixed rapidly, quality hardening, color becomes milky; 4) gonadal tissue sampling procedure: after fixing, 30-60 takes out milky gonadal tissue in second, be placed on and drip in advance on the microslide that has 1 fuchsin acetum, add again 1 crystal violet ethanolic solution by its dyeing 2-4 minute, last covered also flattens gonadal tissue gently, is placed on 4 * 10 biology microscope Microscopic observation;
3) compressing tablet is observed: the sex of differentiating juvenile fish according to the difference of the aspects such as juvenile fish sexual gland shape, cell arrangement mode, cell individual size; Sexual gland shape is comb shape branch; Cell volume is little, and cell is together tightly packed, and cell and cell compartment are very indistinguishable is milter; Sexual gland shape is fiber ligature; Cell volume is relatively large, the rounded or oval of shape, cell arrangement rule, clear-cut be raun.
2. utilize method described in claim 1 to measure a method for hybridization Pelteobagrus fulvidraco male ratio, it is characterized in that carrying out random sampling in ， juvenile fish colony, sample size is more than 100 tails, and the individual body long size of juvenile fish being sampled is more than 3 centimetres; The individuality of sampling is differentiated to sex according to the method for claim 1; According to identification result, draw the male ratio of juvenile fish colony.
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Cited By (2)
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| CN110663617A (en) * | 2019-11-04 | 2020-01-10 | 安徽省农业科学院水产研究所 | Method for identifying separation group of hybrid pelteobagrus fulvidraco in fish breeding stage |
| CN116064759A (en) * | 2022-11-24 | 2023-05-05 | 华中农业大学 | Molecular marker, primer group, kit, method and application for identifying sex of pelteobagrus vachelli |
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Cited By (3)
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| CN110663617A (en) * | 2019-11-04 | 2020-01-10 | 安徽省农业科学院水产研究所 | Method for identifying separation group of hybrid pelteobagrus fulvidraco in fish breeding stage |
| CN116064759A (en) * | 2022-11-24 | 2023-05-05 | 华中农业大学 | Molecular marker, primer group, kit, method and application for identifying sex of pelteobagrus vachelli |
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Application publication date: 20140122 |