CN109874712A - A kind of inseminatio externalis method of litopenaeus vannamei - Google Patents
A kind of inseminatio externalis method of litopenaeus vannamei Download PDFInfo
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Abstract
The invention discloses a kind of inseminatio externalis methods of litopenaeus vannamei, it is the following steps are included: the accelerating the ripening of Litopenaeus vannamei Broodstock, the acquisition of sperm, capacitation, sperm ovum binding and sperm survival rate detecting step, first accelerate the ripening to Litopenaeus vannamei Broodstock, then method is frustrated by using net or pressed disc method obtains sperm, sperm is cultivated in capacitation liquid again can be obtained capacitated sperm, then the capacitated sperm obtained is mixed with ovum, and at the same time carrying out electric shock stimulation, litopenaeus vannamei embryo is obtained finally by water-bath culture.This method is directed to litopenaeus vannamei sperm cell characteristics, selects the sperm of suitable capacitation liquid culture litopenaeus vannamei, improves the fertilizing ability of sperm, simultaneously in Process of Insemination, electric shock stimulation is carried out to smart ovum mixed liquor, promotes the sperm ovum binding of litopenaeus vannamei, promotes artificial insemination effect.
Description
Technical field
The invention belongs to aquaculture raising technology fields, and in particular to a kind of inseminatio externalis method of litopenaeus vannamei.
Background technique
Litopenaeus vannamei (Litopenaeus vannamei), also known as Penaeus Vannmei, the white foot shrimp of alias, Litoenaeus vannamei,
South America Pacific coast waters in source area is suitble in the torrid zone, subtropical zone, warm temperate zone, temperate zone marine site cultivation.Litopenaeus vannamei is
One of big excellent variety of cultured output highest three on our times, it has to water environment high-output stress-resistance, nutritional requirement is low,
The features such as speed of growth is fast, shrimp body meat content is high, out-of-water survival time is long and disease resistance is strong.It is passed through since litopenaeus vannamei cultivates
Ji remarkable benefit, has become and has cultivated the highest prawn kind of largest yield in the world.
During the seed breeding of litopenaeus vannamei, mainly inseminated using spermatophore transplanting and external sperm ovum binding artificial
The spermatophore of male shrimp is adhered to since litopenaeus vannamei belongs to open receptaculum seminis type using spermatophore transplanting mode by insemination method
Easily fallen off when on the receptaculum seminis position of female shrimp, not only technical difficulty of operation is big, but also averagely insemination rate it is low cause it is artificial
Insemination effect is bad, so that subsequent hatching larvae and seed breeding are influenced, so the artificial insemination of external sperm ovum binding insemination is more
It is suitble to the seed breeding of litopenaeus vannamei.With the development of artificial insemination and technology in vitro fertilization, the sperm product of litopenaeus vannamei
Matter becomes an important factor for influencing its seed breeding efficiency, by using suitable capacitation method, when extension sperm survives
Between, sperm fertilizing ability is improved, but there are no the capacitation side that complete set is effectively suitble to litopenaeus vannamei at this stage
Method.
Simultaneously in the sperm ovum binding the time of fertilization for carrying out litopenaeus vannamei, because the sperm of litopenaeus vannamei has atrichia knot
Structure lacks locomitivity and cannot increase the difficulty of sperm ovum binding actively close to ovum, influences the insemination effect of litopenaeus vannamei
Rate realizes the extensive family of litopenaeus vannamei so studying and improving there is still a need for the artificial fertilization technology to litopenaeus vannamei
It is selection and use and cultivation.
Summary of the invention
In view of the above deficiencies, the present invention provides a kind of inseminatio externalis methods of litopenaeus vannamei, for litopenaeus vannamei
Sperm cell characteristics select the sperm of suitable capacitation liquid culture litopenaeus vannamei, improve the fertilizing ability of sperm, while inseminating
Cheng Zhong carries out electric shock stimulation to smart ovum mixed liquor, promotes the sperm ovum binding of litopenaeus vannamei, promotes artificial insemination effect.
The present invention is achieved by the following technical scheme:
A kind of inseminatio externalis method of litopenaeus vannamei comprising following steps:
(1) Litopenaeus vannamei Broodstock accelerates the ripening: by Litopenaeus vannamei Broodstock by 13 tails/m2Cultivation density cultivated, water level
60 cm, water temperature gradually rise to 27~28 DEG C from 22 DEG C of heating rates according to 1 DEG C/min, and cultivation starts with conventional close shrimp
Clinker is fed 3 days, is then fed any one or a few mixing in new Fresh squid, oyster and green worm as biological feed all
Receive shore prawn；Close shrimp ponds soil pick-up is carried out daily, and after draining and reducing 5cm to water level, then moisturizing is to former height of water level；Add
Enter 1ppm probiotics regulation water quality, 0.5~1ppm splashes excellent happy iodine disinfection and sterilization, splash 0.5ppm nutrition calcium and 1ppm amino Portugal
Grape Calciofon；Eyestalk ablation operation, 5 days after operation on the right side of female shrimp is carried out after close shrimp completes first time shell within reinforced cultivating 20 days to open
Begin to carry out semen collection operation；
(2) acquisition of sperm: method is frustrated using net or pressed disc method carries out the acquisition of sperm；The net method of frustrating is by litopenaeus vannamei
Spermatophore be cut into segment and be placed in the screen cloth of 150 mesh, seawater is added dropwise, while gently rubbing spermatophore across screen cloth, to obtain
Obtain sperm suspension；The pressed disc method is to squeeze out the spermatium of litopenaeus vannamei from spermatophore, is then pressed from both sides with two glass slides
Firmly spermatium is coated on sperm on glass slide, then uses seawater flushing glass slide, and collecting washing lotion in revolving speed is 500r/min's
Under the conditions of be centrifuged 10min, collect supernatant, then be centrifuged 5min under conditions of revolving speed is 2000r/min, remove supernatant
Obtain sperm；
(3) capacitation: the sperm suspension obtained in step (2) or sperm are mixed with isometric capacitation liquid, then 30
The mixed liquor A containing capacitated sperm can be obtained in water-bath culture 3h at DEG C；
(4) smart ovum is bathed altogether: being chosen the unfertilized ovum being just discharged of litopenaeus vannamei and is mixed with mixed liquor A obtained in step (3), so
Moment electric shock stimulation is carried out with gene importing equipment afterwards, and can be obtained vannamei boone pair after cultivating 30min in water-bath at 30 DEG C
The embryo of shrimp；
(5) sperm survival rate detects:
A. it takes the Yihong 0.25g to be dissolved in 50 ml seawater, 1ml concentrated hydrochloric acid is then added, stand 12 hours, then filter, incited somebody to action
Screening is placed dries to obtain dried object in oven, and then dried object is dissolved in the ethyl alcohol that 100ml mass concentration is 95% and obtains her
Hongyuan liquid is spare；
B. it takes Yihong stoste that the ethyl alcohol that the mass concentration of 1~2 times of volume is 95% is added and obtains eosin stain, take in step (3)
Mixed liquor A, by mixed liquor A: eosin stain is (1~5): 1 volume ratio mixes mixed liquor A and eosin stain, immediately after
Smear dries observation.
Further, it is 250v that the stimulation of electric shock described in step (4), which is in voltage, and resistance is 500 Ω, and capacitor is 500u's
Under the conditions of carry out.
Further, capacitation liquid described in step (3) is any one in BO liquid, BWW liquid, mTBM liquid and Hank ' s liquid
Kind；
The BO liquid is first by 6.5453g NaCl, 0.2997g KCl, 0.1145g NaH2PO4•2H2O, 0.1057g
MgCl2•6H2O, 0.33g CaCl2, 2.518g glucose, phenol red, 10,000 IU of penicillin that 4mL mass concentration is 0.2%, streptomysin
10000 IU, are dissolved in the seawater of 100mL, then obtain BO storing liquid with 0.22 μm of filter filtration sterilization；By 0.3108g
NaHCO3, 0.0138g Sodium Pyruvate, addition dissolved into 10mL BO storing liquid, with seawater constant volume to 100mL obtain BO work
Liquid；It prepares to obtain BO liquid in the ratio that 20 μ g heparin, 20mg bovine serum albumin(BSA) are added in 1mL BO working solution；
The BWW liquid is first by 5.54g NaCl, 0.356g KCl, 0.25g CaCl2•2H2O, 0.162g KH2PO4,
0.294g MgSO4•7H2O is dissolved in the seawater of 1000mL and obtains BWW stock solution；By 0.21g NaHCO3, 0.37mL mass is dense
The sodium lactate that degree is 60%, 0.003g Sodium Pyruvate, 0.1g glucose, 100,000 U/mL of penicillin, each 100,000 U/mL of streptomysin,
0.477g 4- hydroxyethyl piperazineethanesulfonic acid is added into 100mLBWW stock solution dissolution and obtains BWW working solution, then presses 1mL
5mg bovine serum albumin(BSA), which is added, in BWW working solution can be obtained BWW liquid；
The mTBM liquid is first by 0.6610 g NaCl, 0.0224 g KCl, 0.1103 g CaCl2•2H2O、0.2422 g
Trishydroxymethylaminomethane, 0.1982 g glucose, 0.055 g Sodium Pyruvate, 100 μ L are phenol red, 0.0065 g penicillin,
0.005 g streptomysin, is dissolved in the seawater of 100mL, pH be 7.6~7.8, balance 12 hours after add 10 mg heparin and
MTBM liquid can be obtained in 200m g bovine serum albumin(BSA)；
Hank ' the s liquid is to dissolve 0.8g NaCl, 0.04g KCl, 0.035g NaHCO with 100mL seawater3, 0.016g
CaCl2•2H2O, 0.02g MgSO4•7H2O, 0.006g KH2PO4, 0.006g Na2HPO4, 0.1g glucose obtains.
Further, above-mentioned seawater is the seawater obtained after conventional ultraviolet sterilizer sterilizing.
The technical program has the advantages that compared with prior art
1, capacitation of the BO liquid, BWW liquid, mTBM liquid and Hank ' s liquid that the present invention uses seawater to prepare as litopenaeus vannamei sperm
Liquid promotes capacitation, improves the survival rate and rate of fertilization of sperm；On the other hand since the sperm physiology of litopenaeus vannamei permeates
Pressure is higher, and the capacitation liquid that the present invention uses can be particularly suited for litopenaeus vannamei sperm to avoid spermatogenesis osmotic injury
Culture.
2, the present invention be directed to litopenaeus vannamei physiological make-up, sperm acquisition methods are optimized, using net frustrate method and
Pressed disc method obtains sperm, can effectively improve the pick-up rate and survival rate of sperm.
3, the present invention is directed to the characteristic of litopenaeus vannamei sperm, prepares survey of the suitable eosin stain for sperm survival rate
It is fixed, while sperm suspension and eosin stain mixed proportion are controlled, guarantee the accuracy of sperm survival rate measurement.
4, the present invention be directed to the deficiencies in the prior art, in conjunction with the accelerating the ripening of Litopenaeus vannamei Broodstock, the acquisition of sperm, sperm
Capacitation and sperm survival rate detection and etc., propose complete set effectively be suitble to litopenaeus vannamei capacitation method, prolong
Long sperm improves sperm fertilizing ability at live time, and the seed breeding for subsequent litopenaeus vannamei provides technical support.
5, the sperm of litopenaeus vannamei is first carried out capacitation by the present invention, then it is bathed altogether with the ovum being just discharged and into
Row electric shock stimulation is conducive to sperm and ovum and disperses in seawater, promotes sperm ovum binding, promotes the artificial insemination effect of litopenaeus vannamei
Fruit.
Specific embodiment
The present invention is further illustrated by the following examples, but not as limitation of the present invention.In the following example not
Dated specific experiment condition and method, used technological means are usually conventional hand well-known to those skilled in the art
Section.
Embodiment 1:
A kind of litopenaeus vannamei capacitation method comprising following steps:
(1) Litopenaeus vannamei Broodstock accelerates the ripening: by Litopenaeus vannamei Broodstock by 13 tails/m2Cultivation density cultivated, water level
60 cm, water temperature gradually rise to 27~28 DEG C from 22 DEG C of heating rates according to 1 DEG C/min, and cultivation starts with conventional close shrimp
Clinker is fed 3 days, is then fed any one or a few mixing in new Fresh squid, oyster and green worm as biological feed all
Receive shore prawn；Close shrimp ponds soil pick-up is carried out daily, and after draining and reducing 5cm to water level, then moisturizing is to former height of water level；Add
Enter 1ppm probiotics regulation water quality, 0.5ppm splashes excellent happy iodine disinfection and sterilization, splash 0.5ppm nutrition calcium and 1ppm aminoglucose
Calciofon；Eyestalk ablation operation, 5 days after operation on the right side of female shrimp is carried out after close shrimp completes first time shell within reinforced cultivating 20 days to start
Carry out semen collection operation；
(2) acquisition that method carries out sperm the acquisition of sperm: is frustrated using net；The net method of frustrating is to cut the spermatophore of litopenaeus vannamei
It is placed at segment in the screen cloth of 150 mesh, seawater is added dropwise, while gently rubbing spermatophore across screen cloth, so that it is outstanding to obtain sperm
Liquid；
(3) capacitation: the sperm suspension obtained in step (2) or sperm are mixed with isometric capacitation liquid, then 30
The mixed liquor A containing capacitated sperm can be obtained in water-bath culture 3h at DEG C；The capacitation liquid is BO liquid, and preparation method is first
By 6.5453g NaCl, 0.2997g KCl, 0.1145g NaH2PO4•2H2O, 0.1057g MgCl2•6H2O, 0.33g CaCl2,
2.518g glucose, phenol red, 10,000 IU of penicillin that 4mL mass concentration is 0.2%, 10,000 IU of streptomysin are dissolved in the seawater of 100mL
In, then BO storing liquid is obtained with 0.22 μm of filter filtration sterilization；By 0.3108g NaHCO3, 0.0138g Sodium Pyruvate adds
Enter into 10mL BO storing liquid and dissolve, obtains BO working solution with seawater constant volume to 100mL；By 20 μ are added in 1mL BO working solution
G heparin, 20mg bovine serum albumin(BSA) ratio prepare to obtain BO liquid；
(4) smart ovum is bathed altogether: being chosen the unfertilized ovum being just discharged of litopenaeus vannamei and is mixed with mixed liquor A obtained in step (3), so
Moment electric shock stimulation is carried out with gene importing equipment afterwards, and can be obtained vannamei boone pair after cultivating 30min in water-bath at 30 DEG C
The embryo of shrimp；It is 250v that the electric shock stimulation, which is in voltage, and resistance is 500 Ω, and capacitor carries out under conditions of being 500u；
(5) sperm survival rate detects:
A. it takes the Yihong 0.25g to be dissolved in 50 ml seawater, 1ml concentrated hydrochloric acid is then added, stand 12 hours, then filter, incited somebody to action
Screening is placed dries to obtain dried object in oven, and then dried object is dissolved in the ethyl alcohol that 100ml mass concentration is 95% and obtains her
Hongyuan liquid is spare；
B. it takes Yihong stoste that the ethyl alcohol that the mass concentration of 1 times of volume is 95% is added and obtains eosin stain, take mixed in step (3)
Liquid A is closed, by mixed liquor A: the volume ratio that eosin stain is 4:1 mixes mixed liquor A and eosin stain, and smear dries immediately after
Observation.
Above-mentioned seawater is the seawater obtained after conventional ultraviolet sterilizer sterilizing.
Embodiment 2:
A kind of litopenaeus vannamei capacitation method comprising following steps:
(1) Litopenaeus vannamei Broodstock accelerates the ripening: by Litopenaeus vannamei Broodstock by 13 tails/m2Cultivation density cultivated, water level
60 cm, water temperature gradually rise to 27~28 DEG C from 22 DEG C of heating rates according to 1 DEG C/min, and cultivation starts with conventional close shrimp
Clinker is fed 3 days, is then fed any one or a few mixing in new Fresh squid, oyster and green worm as biological feed all
Receive shore prawn；Close shrimp ponds soil pick-up is carried out daily, and after draining and reducing 5cm to water level, then moisturizing is to former height of water level；Add
Enter 1ppm probiotics regulation water quality, 1ppm splashes excellent happy iodine disinfection and sterilization, splash 0.5ppm nutrition calcium and 1ppm Glucosamine
Sour calcium；Reinforced cultivating 20 days carries out Eyestalk ablation operation on the right side of female shrimp after close shrimp completes first time shell, 5 days after operation start into
The operation of row semen collection；
(2) acquisition of sperm the acquisition of sperm: is carried out using pressed disc method；The pressed disc method is by the spermatium of litopenaeus vannamei
It is squeezed out from spermatophore, then clamps spermatium with two glass slides, be coated on sperm on glass slide, then carried with seawater flushing
Slide collects washing lotion and is centrifuged 10min under conditions of revolving speed is 500r/min, collects supernatant, then in revolving speed is 2000r/min
Under conditions of be centrifuged 5min, removing supernatant can be obtained sperm；
(3) capacitation: the sperm suspension obtained in step (2) or sperm are mixed with isometric capacitation liquid, then 30
The mixed liquor A containing capacitated sperm can be obtained in water-bath culture 3h at DEG C；The capacitation liquid is BWW liquid, and preparation method is
First by 5.54g NaCl, 0.356g KCl, 0.25g CaCl2•2H2O, 0.162g KH2PO4, 0.294g MgSO4•7H2O is dissolved in
BWW stock solution is obtained in the seawater of 1000mL；By 0.21g NaHCO3, the sodium lactate that 0.37mL mass concentration is 60%, 0.003g
Sodium Pyruvate, 0.1g glucose, 100,000 U/mL of penicillin, streptomysin each 100,000 U/mL, 0.477g 4- hydroxyethyl piperazine second sulphur
Acid is added into 100mLBWW stock solution dissolution and obtains BWW working solution, and it is pure that 5mg ox blood then is added by 1mL BWW working solution
BWW liquid can be obtained in albumen；
(4) smart ovum is bathed altogether: being chosen the unfertilized ovum being just discharged of litopenaeus vannamei and is mixed with mixed liquor A obtained in step (3), so
Moment electric shock stimulation is carried out with gene importing equipment afterwards, and can be obtained vannamei boone pair after cultivating 30min in water-bath at 30 DEG C
The embryo of shrimp；It is 250v that the electric shock stimulation, which is in voltage, and resistance is 500 Ω, and capacitor carries out under conditions of being 500u；
(5) sperm survival rate detects:
A. it takes the Yihong 0.25g to be dissolved in 50 ml seawater, 1ml concentrated hydrochloric acid is then added, stand 12 hours, then filter, incited somebody to action
Screening is placed dries to obtain dried object in oven, and then dried object is dissolved in the ethyl alcohol that 100ml mass concentration is 95% and obtains her
Hongyuan liquid is spare；
B. it takes Yihong stoste that the ethyl alcohol that the mass concentration of 2 times of volumes is 95% is added and obtains eosin stain, take mixed in step (3)
Liquid A is closed, by mixed liquor A: the volume ratio that eosin stain is 3:1 mixes mixed liquor A and eosin stain, and smear dries immediately after
Observation.
Above-mentioned seawater is the seawater obtained after conventional ultraviolet sterilizer sterilizing.
Embodiment 3:
A kind of litopenaeus vannamei capacitation method comprising following steps:
(1) Litopenaeus vannamei Broodstock accelerates the ripening: by Litopenaeus vannamei Broodstock by 13 tails/m2Cultivation density cultivated, water level
60 cm, water temperature gradually rise to 27~28 DEG C from 22 DEG C of heating rates according to 1 DEG C/min, and cultivation starts with conventional close shrimp
Clinker is fed 3 days, is then fed any one or a few mixing in new Fresh squid, oyster and green worm as biological feed all
Receive shore prawn；Close shrimp ponds soil pick-up is carried out daily, and after draining and reducing 5cm to water level, then moisturizing is to former height of water level；Add
Enter 1ppm probiotics regulation water quality, 0.8ppm splashes excellent happy iodine disinfection and sterilization, splash 0.5ppm nutrition calcium and 1ppm aminoglucose
Calciofon；Eyestalk ablation operation, 5 days after operation on the right side of female shrimp is carried out after close shrimp completes first time shell within reinforced cultivating 20 days to start
Carry out semen collection operation；
(2) acquisition of sperm the acquisition of sperm: is carried out using pressed disc method；The pressed disc method is by the spermatium of litopenaeus vannamei
It is squeezed out from spermatophore, then clamps spermatium with two glass slides, be coated on sperm on glass slide, then carried with seawater flushing
Slide collects washing lotion and is centrifuged 10min under conditions of revolving speed is 500r/min, collects supernatant, then in revolving speed is 2000r/min
Under conditions of be centrifuged 5min, removing supernatant can be obtained sperm；
(3) capacitation: the sperm suspension obtained in step (2) or sperm are mixed with isometric capacitation liquid, then 30
The mixed liquor A containing capacitated sperm can be obtained in water-bath culture 3h at DEG C；The capacitation liquid is mTBM liquid, and preparation method is
First by 0.6610 g NaCl, 0.0224 g KCl, 0.1103 g CaCl2•2H2O, 0.2422 g trishydroxymethylaminomethane,
0.1982 g glucose, 0.055 g Sodium Pyruvate, 100 μ L are phenol red, 0.0065 g penicillin, 0.005 g streptomysin, molten
In the seawater of 100mL, pH is 7.6~7.8, and balance added 10 mg heparin and 200m g bovine serum albumin(BSA) after 12 hours
MTBM liquid can be obtained；
(4) smart ovum is bathed altogether: being chosen the unfertilized ovum being just discharged of litopenaeus vannamei and is mixed with mixed liquor A obtained in step (3), so
Moment electric shock stimulation is carried out with gene importing equipment afterwards, and can be obtained vannamei boone pair after cultivating 30min in water-bath at 30 DEG C
The embryo of shrimp；It is 250v that the electric shock stimulation, which is in voltage, and resistance is 500 Ω, and capacitor carries out under conditions of being 500u；
(5) sperm survival rate detects:
A. it takes the Yihong 0.25g to be dissolved in 50 ml seawater, 1ml concentrated hydrochloric acid is then added, stand 12 hours, then filter, incited somebody to action
Screening is placed dries to obtain dried object in oven, and then dried object is dissolved in the ethyl alcohol that 100ml mass concentration is 95% and obtains her
Hongyuan liquid is spare；
B. it takes Yihong stoste that the ethyl alcohol that the mass concentration of 1.5 times of volumes is 95% is added and obtains eosin stain, take in step (3)
Mixed liquor A, by mixed liquor A: the volume ratio that eosin stain is 1:1 mixes mixed liquor A and eosin stain, and smear dries in the air immediately after
Dry observation.
Above-mentioned seawater is the seawater obtained after conventional ultraviolet sterilizer sterilizing.
Embodiment 4:
A kind of litopenaeus vannamei capacitation method comprising following steps:
(1) Litopenaeus vannamei Broodstock accelerates the ripening: by Litopenaeus vannamei Broodstock by 13 tails/m2Cultivation density cultivated, water level
60 cm, water temperature gradually rise to 27~28 DEG C from 22 DEG C of heating rates according to 1 DEG C/min, and cultivation starts with conventional close shrimp
Clinker is fed 3 days, is then fed any one or a few mixing in new Fresh squid, oyster and green worm as biological feed all
Receive shore prawn；Close shrimp ponds soil pick-up is carried out daily, and after draining and reducing 5cm to water level, then moisturizing is to former height of water level；Add
Enter 1ppm probiotics regulation water quality, 0.7ppm splashes excellent happy iodine disinfection and sterilization, splash 0.5ppm nutrition calcium and 1ppm aminoglucose
Calciofon；Eyestalk ablation operation, 5 days after operation on the right side of female shrimp is carried out after close shrimp completes first time shell within reinforced cultivating 20 days to start
Carry out semen collection operation；
(2) acquisition that method carries out sperm the acquisition of sperm: is frustrated using net；The net method of frustrating is to cut the spermatophore of litopenaeus vannamei
It is placed at segment in the screen cloth of 150 mesh, seawater is added dropwise, while gently rubbing spermatophore across screen cloth, so that it is outstanding to obtain sperm
Liquid；
(3) capacitation: the sperm suspension obtained in step (2) or sperm are mixed with isometric capacitation liquid, then 30
The mixed liquor A containing capacitated sperm can be obtained in water-bath culture 3h at DEG C；The capacitation liquid is Hank ' s liquid, preparation method
It is to dissolve 0.8g NaCl, 0.04g KCl, 0.035g NaHCO with 100mL seawater3, 0.016g CaCl2•2H2O, 0.02g
MgSO4•7H2O, 0.006g KH2PO4, 0.006g Na2HPO4, 0.1g glucose obtains；
(4) smart ovum is bathed altogether: being chosen the unfertilized ovum being just discharged of litopenaeus vannamei and is mixed with mixed liquor A obtained in step (3), so
Moment electric shock stimulation is carried out with gene importing equipment afterwards, and can be obtained vannamei boone pair after cultivating 30min in water-bath at 30 DEG C
The embryo of shrimp；It is 250v that the electric shock stimulation, which is in voltage, and resistance is 500 Ω, and capacitor carries out under conditions of being 500u；
(5) sperm survival rate detects:
A. it takes the Yihong 0.25g to be dissolved in 50 ml seawater, 1ml concentrated hydrochloric acid is then added, stand 12 hours, then filter, incited somebody to action
Screening is placed dries to obtain dried object in oven, and then dried object is dissolved in the ethyl alcohol that 100ml mass concentration is 95% and obtains her
Hongyuan liquid is spare；
B. it takes Yihong stoste that the ethyl alcohol that the mass concentration of 1 times of volume is 95% is added and obtains eosin stain, take mixed in step (3)
Liquid A is closed, by mixed liquor A: the volume ratio that eosin stain is 2:1 mixes mixed liquor A and eosin stain, and smear dries immediately after
Observation.
Above-mentioned seawater is the seawater obtained after conventional ultraviolet sterilizer sterilizing.
Embodiment 5:
A kind of litopenaeus vannamei capacitation method comprising following steps:
(1) Litopenaeus vannamei Broodstock accelerates the ripening: by Litopenaeus vannamei Broodstock by 13 tails/m2Cultivation density cultivated, water level
60 cm, water temperature gradually rise to 27~28 DEG C from 22 DEG C of heating rates according to 1 DEG C/min, and cultivation starts with conventional close shrimp
Clinker is fed 3 days, is then fed any one or a few mixing in new Fresh squid, oyster and green worm as biological feed all
Receive shore prawn；Close shrimp ponds soil pick-up is carried out daily, and after draining and reducing 5cm to water level, then moisturizing is to former height of water level；Add
Enter 1ppm probiotics regulation water quality, 0.6ppm splashes excellent happy iodine disinfection and sterilization, splash 0.5ppm nutrition calcium and 1ppm aminoglucose
Calciofon；Eyestalk ablation operation, 5 days after operation on the right side of female shrimp is carried out after close shrimp completes first time shell within reinforced cultivating 20 days to start
Carry out semen collection operation；
(2) acquisition of sperm the acquisition of sperm: is carried out using pressed disc method；The pressed disc method is by the spermatium of litopenaeus vannamei
It is squeezed out from spermatophore, then clamps spermatium with two glass slides, be coated on sperm on glass slide, then carried with seawater flushing
Slide collects washing lotion and is centrifuged 10min under conditions of revolving speed is 500r/min, collects supernatant, then in revolving speed is 2000r/min
Under conditions of be centrifuged 5min, removing supernatant can be obtained sperm；
(3) capacitation: the sperm suspension obtained in step (2) or sperm are mixed with isometric capacitation liquid, then 30
The mixed liquor A containing capacitated sperm can be obtained in water-bath culture 3h at DEG C；The capacitation liquid is BO liquid, and preparation method is first
By 6.5453g NaCl, 0.2997g KCl, 0.1145g NaH2PO4•2H2O, 0.1057g MgCl2•6H2O, 0.33g CaCl2,
2.518g glucose, phenol red, 10,000 IU of penicillin that 4mL mass concentration is 0.2%, 10,000 IU of streptomysin are dissolved in the seawater of 100mL
In, then BO storing liquid is obtained with 0.22 μm of filter filtration sterilization；By 0.3108g NaHCO3, 0.0138g Sodium Pyruvate adds
Enter into 10mL BO storing liquid and dissolve, obtains BO working solution with seawater constant volume to 100mL；By 20 μ are added in 1mL BO working solution
G heparin, 20mg bovine serum albumin(BSA) ratio prepare to obtain BO liquid；
(4) smart ovum is bathed altogether: being chosen the unfertilized ovum being just discharged of litopenaeus vannamei and is mixed with above-mentioned mixed liquid B, then uses gene
Introducing apparatus carries out moment electric shock stimulation, and can be obtained the embryo of litopenaeus vannamei in water-bath after culture 30min at 30 DEG C；
It is 250v that the electric shock stimulation, which is in voltage, and resistance is 500 Ω, and capacitor carries out under conditions of being 500u；
(5) sperm survival rate detects:
C. it takes the Yihong 0.25g to be dissolved in 50 ml seawater, 1ml concentrated hydrochloric acid is then added, stand 12 hours, then filter, incited somebody to action
Screening is placed dries to obtain dried object in oven, and then dried object is dissolved in the ethyl alcohol that 100ml mass concentration is 95% and obtains her
Hongyuan liquid is spare；
D. it takes Yihong stoste that the ethyl alcohol that the mass concentration of 2 times of volumes is 95% is added and obtains eosin stain, take mixed in step (3)
Liquid A is closed, by mixed liquor A: the volume ratio that eosin stain is 5:1 mixes mixed liquor A and eosin stain, and smear dries immediately after
Observation.
Above-mentioned seawater is the seawater obtained after conventional ultraviolet sterilizer sterilizing.
Comparative example 1:
Use Fish Ringer ' s liquid as capacitation liquid, the body of litopenaeus vannamei is then carried out according to method described in embodiment 1
Outer insemination operation；Fish Ringer ' the s liquid is to dissolve 1.35g NaCl, 0.06g KCl, 0.025g with 100mL seawater
CaCl2, 0.035g MgCl2, 0.02g NaHCO3, 0.03g NaH2PO4It obtains afterwards.
Comparative example 2:
Use Mounib ' s liquid as capacitation liquid, the sperm body of litopenaeus vannamei is then carried out according to method as described in example 2
Outer insemination operation；Mounib ' the s liquid is to dissolve 0.439g NaCl, 0.521g KCl, 0.022g with 100mL seawater
CaCl2, 0.012g MgSO4, obtain after 0.655g Tris.
The inseminatio externalis operation of litopenaeus vannamei, inspection are carried out according to method described in Examples 1 to 5 and comparative example 1~2
Survival rate after surveying capacitation, while using the acrosome reaction of CBB dyeing detection method detection capacitated sperm, statistics acrosome is anti-
It should rate；The concrete outcome that above-mentioned experiment obtains is shown in Table 1.
The survival rate and acrosome reaction rate of 1 capacitated sperm of table
By above-mentioned experimental data it is found that using the Examples 1 to 5 of the method for the present invention compared with comparative example 1~2, although sperm
Survival rate after capacitation is declined, but acrosome reaction rate greatly improved, because acrosome reaction is sent out after capacitation
Raw important physiology course is the prerequisite of fertilization, and only the sperm of completion acrosome reaction could be with oocyte fusion reality
It is now fertilized, so can be improved the acrosome reaction rate of litopenaeus vannamei sperm using the method for the present invention, promotes sperm ovum binding, into one
Step improves artificial insemination efficiency.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (4)
1. a kind of inseminatio externalis method of litopenaeus vannamei, it is characterised in that: itself the following steps are included:
Litopenaeus vannamei Broodstock accelerates the ripening: by Litopenaeus vannamei Broodstock by 13 tails/m2Cultivation density cultivated, water level 60
Cm, water temperature gradually rise to 27~28 DEG C from 22 DEG C of heating rates according to 1 DEG C/min, and cultivation starts with conventional close shrimp clinker
It feeds 3 days, any one or a few mixing in new Fresh squid, oyster and green worm is then fed into vannamei boone as biological feed
Prawn；Close shrimp ponds soil pick-up is carried out daily, and after draining and reducing 5cm to water level, then moisturizing is to former height of water level；It is added
1ppm probiotics regulates and controls water quality, the excellent happy iodine disinfection and sterilization of the 0.5~1ppm that splashes, splash 0.5ppm nutrition calcium and 1ppm aminoglucose
Calciofon；Eyestalk ablation operation, 5 days after operation on the right side of female shrimp is carried out after close shrimp completes first time shell within reinforced cultivating 20 days to start
Carry out semen collection operation；
The acquisition of sperm: method is frustrated using net or pressed disc method carries out the acquisition of sperm；The net method of frustrating is by litopenaeus vannamei
Spermatophore is cut into segment and is placed in the screen cloth of 150 mesh, seawater is added dropwise, while gently rubbing spermatophore across screen cloth, to obtain
Sperm suspension；The pressed disc method is to squeeze out the spermatium of litopenaeus vannamei from spermatophore, is then clamped with two glass slides
Spermatium is coated on sperm on glass slide, then uses seawater flushing glass slide, collects washing lotion in the item that revolving speed is 500r/min
It is centrifuged 10min under part, collects supernatant, then be centrifuged 5min under conditions of revolving speed is 2000r/min, removing supernatant can obtain
Obtain sperm；
Capacitation: the sperm suspension obtained in step (2) or sperm are mixed with isometric capacitation liquid, then at 30 DEG C
The mixed liquor A containing capacitated sperm can be obtained in water-bath culture 3h；
Smart ovum is bathed altogether: being chosen the unfertilized ovum being just discharged of litopenaeus vannamei and is mixed with mixed liquor A obtained in step (3), then
Moment electric shock stimulation is carried out with gene importing equipment, and can be obtained litopenaeus vannamei after cultivating 30min in water-bath at 30 DEG C
Embryo；
Sperm survival rate detection:
It takes the Yihong 0.25g to be dissolved in 50 ml seawater, 1ml concentrated hydrochloric acid is then added, stand 12 hours, then filter, will filter
Object is placed to be dried to obtain dried object in oven, and then dried object is dissolved in the ethyl alcohol that 100ml mass concentration is 95% and obtains Yihong
Stoste is spare；
It takes Yihong stoste that the ethyl alcohol that the mass concentration of 1~2 times of volume is 95% is added and obtains eosin stain, take mixed in step (3)
Liquid A is closed, by mixed liquor A: eosin stain is (1~5): 1 volume ratio mixes mixed liquor A and eosin stain, applies immediately after
Piece dries observation.
2. according to the inseminatio externalis method of litopenaeus vannamei described in claim 1, it is characterised in that: the capacitation liquid is BO
Any one in liquid, BWW liquid, mTBM liquid and Hank ' s liquid；
The BO liquid is first by 6.5453g NaCl, 0.2997g KCl, 0.1145g NaH2PO4•2H2O, 0.1057g
MgCl2•6H2O, 0.33g CaCl2, 2.518g glucose, phenol red, 10,000 IU of penicillin that 4mL mass concentration is 0.2%, streptomysin
10000 IU, are dissolved in the seawater of 100mL, then obtain BO storing liquid with 0.22 μm of filter filtration sterilization；By 0.3108g
NaHCO3, 0.0138g Sodium Pyruvate, addition dissolved into 10mL BO storing liquid, with seawater constant volume to 100mL obtain BO work
Liquid；It prepares to obtain BO liquid in the ratio that 20 μ g heparin, 20mg bovine serum albumin(BSA) are added in 1mL BO working solution；
The BWW liquid is first by 5.54g NaCl, 0.356g KCl, 0.25g CaCl2•2H2O, 0.162g KH2PO4,
0.294g MgSO4•7H2O is dissolved in the seawater of 1000mL and obtains BWW stock solution；By 0.21g NaHCO3, 0.37mL mass is dense
The sodium lactate that degree is 60%, 0.003g Sodium Pyruvate, 0.1g glucose, 100,000 U/mL of penicillin, each 100,000 U/mL of streptomysin,
0.477g 4- hydroxyethyl piperazineethanesulfonic acid is added into 100mLBWW stock solution dissolution and obtains BWW working solution, then presses 1mL
5mg bovine serum albumin(BSA), which is added, in BWW working solution can be obtained BWW liquid；
The mTBM liquid is first by 0.6610 g NaCl, 0.0224 g KCl, 0.1103 g CaCl2•2H2O、0.2422 g
Trishydroxymethylaminomethane, 0.1982 g glucose, 0.055 g Sodium Pyruvate, 100 μ L are phenol red, 0.0065 g penicillin,
0.005 g streptomysin, is dissolved in the seawater of 100mL, pH be 7.6~7.8, balance 12 hours after add 10 mg heparin and
MTBM liquid can be obtained in 200m g bovine serum albumin(BSA)；
Hank ' the s liquid is to dissolve 0.8g NaCl, 0.04g KCl, 0.035g NaHCO with 100mL seawater3, 0.016g
CaCl2•2H2O, 0.02g MgSO4•7H2O, 0.006g KH2PO4, 0.006g Na2HPO4, 0.1g glucose obtains.
3. the inseminatio externalis method of litopenaeus vannamei according to claim 1, it is characterised in that: the electric shock, which stimulates, is
It is 250v in voltage, resistance is 500 Ω, and capacitor carries out under conditions of being 500u.
4. the inseminatio externalis method of litopenaeus vannamei according to claim 1, it is characterised in that: the seawater be by
The seawater obtained after conventional ultraviolet sterilizer sterilizing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112753632A (en) * | 2021-01-15 | 2021-05-07 | 广西壮族自治区水产科学研究院 | Method for cutting eye handle of breeding shrimp |
| CN115005134A (en) * | 2022-06-14 | 2022-09-06 | 海南省菜篮农业与渔业发展有限公司 | In-vitro hatching method for freshwater shrimps |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1761131A1 (en) * | 1990-05-28 | 1992-09-15 | Днепропетровский Филиал Украинского Научно-Исследовательского Института Разведения И Искусственного Осеменения Крупного Рогатого Скота | Method for electrostimulation of the genital system of sire bulls |
| RU1813423C (en) * | 1990-11-22 | 1993-05-07 | Научно-Производственное И Информационное Объединение "Биоиндустрия" | Method for detection of estrus in animals |
| CN1385534A (en) * | 2001-05-16 | 2002-12-18 | 中国科学院水生生物研究所 | Fish electric pulse sperm transgene method |
| CA2865424A1 (en) * | 2011-02-28 | 2012-09-07 | Rigshospitalet Copenhagen University Hospital | Active vitamin d or analogs thereof for use in in vivo or in vitro fertilization |
| CN105532537A (en) * | 2016-01-05 | 2016-05-04 | 中国水产科学研究院淡水渔业研究中心 | Method for extracting macrobrachium spermatophore through pulse electric shock method and macrobrachium intraspecific crossbreeding method |
| CN105594635A (en) * | 2015-12-19 | 2016-05-25 | 太和县顺发养殖有限公司 | Culture method for good-quality and high-yield catfish |
| CN106900617A (en) * | 2015-12-21 | 2017-06-30 | 青岛清泉生物科技有限公司 | The gynogenesis abductive approach of Environment of Litopenaeus vannamei Low |
-
2019
- 2019-03-07 CN CN201910170040.3A patent/CN109874712B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1761131A1 (en) * | 1990-05-28 | 1992-09-15 | Днепропетровский Филиал Украинского Научно-Исследовательского Института Разведения И Искусственного Осеменения Крупного Рогатого Скота | Method for electrostimulation of the genital system of sire bulls |
| RU1813423C (en) * | 1990-11-22 | 1993-05-07 | Научно-Производственное И Информационное Объединение "Биоиндустрия" | Method for detection of estrus in animals |
| CN1385534A (en) * | 2001-05-16 | 2002-12-18 | 中国科学院水生生物研究所 | Fish electric pulse sperm transgene method |
| CA2865424A1 (en) * | 2011-02-28 | 2012-09-07 | Rigshospitalet Copenhagen University Hospital | Active vitamin d or analogs thereof for use in in vivo or in vitro fertilization |
| CN105594635A (en) * | 2015-12-19 | 2016-05-25 | 太和县顺发养殖有限公司 | Culture method for good-quality and high-yield catfish |
| CN106900617A (en) * | 2015-12-21 | 2017-06-30 | 青岛清泉生物科技有限公司 | The gynogenesis abductive approach of Environment of Litopenaeus vannamei Low |
| CN105532537A (en) * | 2016-01-05 | 2016-05-04 | 中国水产科学研究院淡水渔业研究中心 | Method for extracting macrobrachium spermatophore through pulse electric shock method and macrobrachium intraspecific crossbreeding method |
Non-Patent Citations (4)
| Title |
|---|
| ANDRÉ BRAGA ET AL.: "Spermatozoal capacitation of pink shrimp Farfantepenaeus paulensis", 《AQUACULTURE》 * |
| JORGE ALFARO ET AL.: "Sperm maturation and capacitation in the open thelycum shrimp Litopenaeus (Crustacea：Decapoda：Penaeoidea)", 《AQUACULTURE》 * |
| SIRINDA AUNGSUCHAWAN1 ET AL.: "Sperm capacitation of the shrimp Litopenaeus vannamei", 《AQUACULTURE RESEARCH》 * |
| 张伟权 等: "南美洲白对虾全人工授精技术研究", 《海洋和湖沼》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112753632A (en) * | 2021-01-15 | 2021-05-07 | 广西壮族自治区水产科学研究院 | Method for cutting eye handle of breeding shrimp |
| CN115005134A (en) * | 2022-06-14 | 2022-09-06 | 海南省菜篮农业与渔业发展有限公司 | In-vitro hatching method for freshwater shrimps |
| CN115005134B (en) * | 2022-06-14 | 2023-07-28 | 海南省菜篮农业与渔业发展有限公司 | In-vitro hatching method for freshwater shrimps |
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