CN104808005A - Method for identifying male giant grouper - Google Patents

Method for identifying male giant grouper Download PDF

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Publication number
CN104808005A
CN104808005A CN201510179104.8A CN201510179104A CN104808005A CN 104808005 A CN104808005 A CN 104808005A CN 201510179104 A CN201510179104 A CN 201510179104A CN 104808005 A CN104808005 A CN 104808005A
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serum
epinephelus lanceolatus
lanceolatus fish
fish
male
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CN104808005B (en
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张勇
王庆
李水生
王翔
肖玲
彭诚
杨宇晴
张海发
林浩然
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Sun Yat Sen University
National Sun Yat Sen University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
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  • Urology & Nephrology (AREA)
  • Hematology (AREA)
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  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
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  • Analytical Chemistry (AREA)
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  • General Health & Medical Sciences (AREA)
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Abstract

The invention discloses a method for identifying male giant grouper. The method comprises steps as follows: (1) selecting giant grouper: selecting giant grouper in the breeding season; (2) obtaining serum of the giant grouper: selecting caudal vein blood of the giant grouper, and performing centrifugation to obtain the serum; (3) measuring the content of vitellogenin in the serum: measuring the content of the vitellogenin in the serum through ELISA (enzyme-linked immunosorbent assay); (4) measuring the content of 11-KT (11-ketotestosterone) in the serum: measuring the content of the 11-KT with ELISA in the serum; (5) identifying the male giant grouper: identifying the male giant grouper in combination with the content of the vitellogen and the content of the 11-KT in the serum of the giant grouper. The method is quick, efficient and high in accuracy and causes small harm to the giant grouper.

Description

A kind of method differentiating male epinephelus lanceolatus fish
Technical field
The invention belongs to grouper sex abnormality technical field, be specifically related to a kind of method differentiating male epinephelus lanceolatus fish.
Background technology
Epinephelus lanceolatus fish cries again dragon wholesale, and Perciformes, Anabantoidei ， Sushi section, Epinephelinae, Epinephelus is the one that in grouper, build is maximum.Epinephelus lanceolatus fish individuality is large, nutritious, delicious meat, fast growth, is the fish treasure that people like.In recent years because offshore is excessively captured, output falls sharply, and supply falls short of demand in market.Become the main object of sea-farming ground epinephelus lanceolatus fishes such as coastal areas of southern China Guangdong, Hainan, Fujian, Guangxi, economic worth is huge.
The ripe slow and existence reversal of epinephelus lanceolatus piscinity, belongs to protogyny, hermaphroditic fish.Exactly because this characteristic of hermaphroditic, be therefore difficult to when seed selection parent population only pick out male parent population from external appearance characteristic, cause epinephelus lanceolatus fish parent fish to cultivate difficulty for this reason and strengthen, become a difficult problem of epinephelus lanceolatus fish breeding work.
At present, judge male epinephelus lanceolatus fish Main Basis three point: the size at build and age, see gonopore position, directly extract the method for sexual gland.Three kinds of methods respectively have relative merits, and it is convenient to judge according to build and the size at age, and the time is short, simple to operate, but this method accuracy is low, and the subjectivity impact of people is too large, and feeding method simultaneously, growing environment is larger on its impact.See gonopore fast position, simple to operate, to fish fanout free region, but non-reproduction period, the gonopore slight indentation belly that often diminishes was at this time be difficult to judge male and female, and accuracy is low.High by the method judgment accuracy directly extracting sexual gland, but larger to the injury of fish, and affect gonadal maturation and even cause death, simultaneously complex operation, workload is large, and determination time is long.
Vitellogenin (vitellogenin, Vtg) is the special a kind of albumen be present in oviparity non-mammalian sexal maturity animal blood, is the precursor of livetin (Yolk protein).The important physiology courses such as oviparous animal reproduction, growth are participated in as a kind of important reproduction albumen.Vitellogenin is considered to a kind of desirable estrogen and oestrogen-like hormone mark.Usual Vtg can only detect in the raun blood plasma of breeding period, milter and juvenile fish in-vivo content very small.11-ketone group testosterone (11-ketotestosterone, 11-KT) is the important male steroid of one in fish.Be transformed by testosterone, testosterone changes 11p-hydroxytestosterone into, and the latter forms 11-KT by 11beta-Hydroxysteroid dehydrogenase catalysis again, directly induces fish sperm to generate.
In mating period, the content of these two kinds of hormones of male and female fish has larger difference.Therefore, the present invention intends by detecting and comparing the content of these two kinds of hormones in serum and then identify male epinephelus lanceolatus fish.
Summary of the invention
The object of the present invention is to provide a kind of method differentiating male epinephelus lanceolatus fish, rapidly and efficiently, little to the injury of fish, accuracy is high for the method.
Above-mentioned purpose of the present invention can be achieved through the following technical solutions: a kind of method differentiating male epinephelus lanceolatus fish, comprises the following steps:
(1) epinephelus lanceolatus fish is selected: the epinephelus lanceolatus fish in selected reproduction season;
(2) epinephelus lanceolatus fish serum is obtained: choose epinephelus lanceolatus fish tail venous blood, centrifugal rear acquisition serum;
(3) vitellogenin content in serum is measured: adopt the vitellogenin content in enzyme-linked immunosorbent assay serum:
(4) 11-ketone group testosterone concentration in serum is measured: adopt the 11-ketone group testosterone concentration in enzyme-linked immunosorbent assay serum;
(5) epinephelus lanceolatus fish milter is differentiated: in conjunction with vitellogenin content in epinephelus lanceolatus fish serum and 11-ketone group testosterone concentration, identify epinephelus lanceolatus fish milter.
In the method for the male epinephelus lanceolatus fish of above-mentioned discriminating:
In step (1), selective body focuses on the epinephelus lanceolatus fish of the mating period of more than 20kg, and mating period is coastal 5 preferably annual ~ August in south.
Epinephelus lanceolatus fish tail venous blood 2 ~ 5mL is preferably chosen, centrifugal rear acquisition serum in step (2).
Time centrifugal in step (2), centrifuge speed is preferably 3500 ~ 5000rpm, and centrifuging temperature is preferably 4 DEG C, and centrifugation time is preferably 10 ~ 15min.
When adopting the vitellogenin content in enzyme-linked immunosorbent assay serum in step (3), the serum vitellogenin assay kit measurement of preferred employing caymanchemical company, the operation instruction of concrete flow process reference reagent box.
When adopting the 11-ketone group testosterone concentration in enzyme-linked immunosorbent assay serum in step (4), the serum 11-ketone group testosterone concentration of preferred employing caymanchemical company measures kit measurement, the operation instruction of concrete flow process reference reagent box.
In step (5) as vitellogenin content < 0.9mg/mL in epinephelus lanceolatus fish serum and 11-ketone group testosterone concentration > 1ng/mL time, differentiate as epinephelus lanceolatus fish milter.
Tool of the present invention has the following advantages: the inventive method carries out the discriminating of male epinephelus lanceolatus fish by vitellogenin content in Simultaneously test epinephelus lanceolatus fish serum and 11-ketone group testosterone concentration, and accuracy is high, simple, and little to the injury of fish body.
Embodiment
Embodiment 1
A kind of method differentiating epinephelus lanceolatus fish milter that the present embodiment provides, concrete steps are as follows:
(1) the epinephelus lanceolatus fish of more than the 20kg of 30 Dayawan Aquatic Products Experimental Center, Guangdong Prov.'s cultivation is chosen on June 21st, 2014, now from the male and female that can not identify these fishes in appearance, every bar fish dorsal muscle stamps electronic marker, the mark number that the good every bar fish of record is corresponding;
(2) tail venous blood sampling method is adopted, the centrifuge tube of 15mL is put into the blood of 50mL syringe extraction 2mL, the centrifuge tube that blood is housed is placed on 4 DEG C of refrigerator overnight, second day on hydro-extractor 4 DEG C, the centrifugal 15min of rotating speed of 5000 rev/min, the centrifuge tube that absorption supernatant puts into 1.5mL sterilizing is for subsequent use;
(3) adopt the content of serum VTG assay kit measurement VTG in 30 epinephelus lanceolatus fish serums of cayman chemical company, concrete mensuration process is as follows:
(a) dilution Dilution Buffer: get 15mL 5 × Dilution Buffer and add 60mLddH 2o is for subsequent use; WashBuffer: get 1 bottle of Wash Buffer and add 1000mL ddH 2o is for subsequent use; Standard: get 5 μ g Standard powder dissolutions at 1mLddH 2o, then draw with liquid-transfering gun the Standard that 50 μ L previous steps dissolve and add the Dilution Buffer that 4950 μ L have diluted, then half-and-half dilute until concentration becomes 0.05ng/mL; All dilution are all carried out in the centrifuge tube of the appropriate size of sterilizing, and above diluent materials is all that kit carries.
(b) dilute sample: get 10 μ L blood serum samples and add 490 μ LDilution Buffer, get the blood serum sample that 10 μ L previous steps have diluted again and add 990 μ L Dilution Buffer, get the blood serum sample that 10 μ L previous steps have diluted again and add 990 μ l Dilution Buffer, so dilution three concentration;
(c) application of sample: 96 orifice plates taken out in kit have planned blank well, standard sample wells, sample well, add 100 μ LDilution Buffer in blank well, add Standard that 100 μ L have diluted in standard sample wells, add sample that 100 μ L have diluted in sample well, room temperature places 1h, dilutes antibody: 24 μ L antibody are added 12mL Dilution Buffer in the process of placing 1h; 3 times are washed with 300 μ L Wash Buffer after 1h, add antibody that 100 μ L have diluted again in each hole, room temperature washes 5 times with 300 μ LWash Buffer after placing 1h again, then adds 100 μ LTMB (kit carries) in institute is porose, places 15 minutes in dark; Finally add 0.3M sulfuric acid in institute is porose, cessation reaction.
D () reads data in microplate reader, record data.
(4) adopt the content of 11-KT in 11-KT assay kit measurement 30 epinephelus lanceolatus fish serums of cayman chemical company, concrete grammar is as follows:
(a) dilution EIA Buffer: get 10mL EIA Buffer and join 90mL ddH 2in O; Wash Buffer prepares: get 5mLWash Buffer mother liquor and join 1995mLddH 21mL ploysorbate is added again in O; EIA Trace dilutes: get 100dtn EIA Trace and add the EIA Buffer that 6mL diluted; EIA antiserum dilutes: get 100dtn and add the EIA Buffer that 6mL diluted; Standard dilutes: the Standard getting 100 μ L 10ng/mol adds 900 μ LddH 2in O, therefrom taking out 100 μ L again joins in the EIA Buffer that 900 μ L have diluted, get 500 μ L again to join in the EIA Buffer that 500 μ L have diluted, then the 500 μ L getting previous step join the EIABuffer that 500 μ L have diluted, and are diluted to 0.78pg/mL successively; Ellman Reagent dilutes: get 100dnt Ellman Reagent and add 20mLddH 2o; AchE Tracer dilutes: get 100dtn AchE Tracer and add the EIA Buffer that 6mL diluted; All dilution are all carried out in the centrifuge tube of the different size of sterilizing, and above diluent materials is all that kit carries;
The preparation of (b) sample: get in the sterilized centrifuge tube of 100 μ L serum 1.5mL, then draw 5 μ L serum with liquid-transfering gun and join 955 μ L ddH 2in O, then the serum 5 μ L getting a step dilution adds 955ul ddH 2in O, be so diluted to three concentration;
(c) application of sample: draw TA, BLK, NSB, B on 96 orifice plates 0, TAHE and standard items hole, add in 100 μ LEIA Buffer to NSB holes, add EIA Buffer to the B that 50 μ L have diluted 0in, standard items are added by concentration order from big to small, the sample will diluted again, three concentration are divided to add in three parallel holes, add 50 μ L AchE Tracer to each hole except TAHE, BLK, add each hole except TA, NSB and BLK on 50 μ L EIA antiserum to plate, room temperature washes 5 times with wash buffer after placing 2h, add 200 μ L Ellman Reagent in each hole, add 5 μ L AchE tracer to each hole except TA, build film dark place and put 90 ~ 120 minutes.
D () reads data in microplate reader, record data;
(5) according to vitellogenin content <0.9mg/mL in serum and 11-ketone group testosterone concentration >1ng/mL then differentiates as milter standard, discriminating is carried out to the result measured and draws in 30 fishes have 11 milters (table 1);
(6) sexual gland withdrawal device is adopted to extract a small amount of gonadal tissue of the milter that 11 judge by the inventive method, put in Bo Enshi liquid, carry out the accuracy of paraffin-embedded tissue section for verifying the method, when extracting sexual gland, the mark of corresponding good every bar fish is seen, 11 milters that section display the present invention identifies are all correct, and result of determination rate of accuracy reached of the present invention is to 100%.
Table 1 VTG and 11-KT content and the result of determination to Daya Gulf 30 fishes thereof
Embodiment 2
Step is similar to Example 1, its difference is to choose the place of parent population and the time chooses the epinephelus lanceolatus fish of 25 body weight at more than 20kg at Sanya, Hainan on July 15th, 2014, mark, blood 4 DEG C of placements of tail venous puncture 5mL are spent the night, on hydro-extractor 4 DEG C, the centrifugal 12min of the rotating speed of 3500 rev/min obtains serum, and with the content of kit measurement vitellogenin and 11-ketone group testosterone, the result of mensuration draws in 25 fishes have 6 milters (table 2).Judge that 6 milters identified are all correct by extracting sexual gland.
Table 2 VTG and 11-KT content and the result of determination to 25 fishes in Sanya thereof
Table 3 the inventive method with differentiated that epinephelus lanceolatus fish milter method contrasted in the past
To sum up, show that the inventive method has higher accuracy and larger use value by the result of observation sample, synthetic determination rate of accuracy reached is to 100%, the discrimination method of epinephelus lanceolatus fish milter provided by the invention compares with previous methods and has efficiently and accurately, simple, the advantages (table 3) such as little are injured to parent population.This technology will have significant economic benefit and social benefit in fish breeding.
Above-described embodiment is the present invention's preferably embodiment, but embodiments of the present invention are not restricted to the described embodiments.Change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify, all should be the substitute mode of equivalence, be included in protection scope of the present invention.

Claims (6)

1. differentiate a method for male epinephelus lanceolatus fish, it is characterized in that comprising the following steps:
(1) epinephelus lanceolatus fish is selected: the epinephelus lanceolatus fish in selected reproduction season;
(2) epinephelus lanceolatus fish serum is obtained: choose epinephelus lanceolatus fish tail venous blood, centrifugal rear acquisition serum;
(3) vitellogenin content in serum is measured: adopt the vitellogenin content in enzyme-linked immunosorbent assay serum:
(4) 11-ketone group testosterone concentration in serum is measured: adopt the 11-ketone group testosterone concentration in enzyme-linked immunosorbent assay serum;
(5) epinephelus lanceolatus fish milter is differentiated: in conjunction with vitellogenin content in epinephelus lanceolatus fish serum and 11-ketone group testosterone concentration, identify epinephelus lanceolatus fish milter.
2. the method for the male epinephelus lanceolatus fish of discriminating according to claim 1, is characterized in that: in step (1), selective body focuses on the epinephelus lanceolatus fish of the mating period of more than 20kg, and mating period is coastal in south is 5 annual ~ August.
3. the method for the male epinephelus lanceolatus fish of discriminating according to claim 1, it is characterized in that: time centrifugal in step (2), centrifuge speed is 3500 ~ 5000rpm, centrifuging temperature is 4 DEG C, and centrifugation time is 10 ~ 15min.
4. the method for the male epinephelus lanceolatus fish of discriminating according to claim 1, it is characterized in that: when adopting the vitellogenin content in enzyme-linked immunosorbent assay serum in step (3), adopt the serum vitellogenin assay kit measurement of cayman chemical company.
5. the method for the male epinephelus lanceolatus fish of discriminating according to claim 1, it is characterized in that: when adopting the 11-ketone group testosterone concentration in enzyme-linked immunosorbent assay serum in step (4), adopt the serum 11-ketone group testosterone concentration of cayman chemical company to measure kit measurement.
6. the method for the male epinephelus lanceolatus fish of discriminating according to claim 1, it is characterized in that: in step (5) as vitellogenin content < 0.9mg/mL in epinephelus lanceolatus fish serum and 11-ketone group testosterone concentration > 1ng/mL time, differentiate as epinephelus lanceolatus fish milter.
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CN106947816A (en) * 2016-10-28 2017-07-14 中山大学 A kind of method of Epinephelus coioides paternity test microsatellite Multiplex fluorescent PCR
CN111018967A (en) * 2019-12-31 2020-04-17 广东越群海洋生物研究开发有限公司 Epinephelus lanceolatus insulin-like factor INSL-3 gene, encoding protein and application thereof

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CN104285866A (en) * 2014-10-23 2015-01-21 福建省水产研究所 Technique for masculinizing large groupers by surgical implanting

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CN102707073A (en) * 2012-06-21 2012-10-03 江苏大学 Enzyme linked immunosorbent assay kit for detecting metandienone and using method of enzyme linked immunosorbent assay kit
CN104285866A (en) * 2014-10-23 2015-01-21 福建省水产研究所 Technique for masculinizing large groupers by surgical implanting

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106947816A (en) * 2016-10-28 2017-07-14 中山大学 A kind of method of Epinephelus coioides paternity test microsatellite Multiplex fluorescent PCR
CN106947816B (en) * 2016-10-28 2020-08-04 中山大学 Microsatellite fluorescent multiplex PCR (polymerase chain reaction) method for parent-child identification of epinephelus coioides
CN111018967A (en) * 2019-12-31 2020-04-17 广东越群海洋生物研究开发有限公司 Epinephelus lanceolatus insulin-like factor INSL-3 gene, encoding protein and application thereof
CN111018967B (en) * 2019-12-31 2020-09-22 广东越群海洋生物研究开发有限公司 Epinephelus lanceolatus insulin-like factor INSL-3 gene, encoding protein and application thereof

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