CN103891711A - Treatment method for seminal fluid of lateolabrax japonicus - Google Patents
Treatment method for seminal fluid of lateolabrax japonicus Download PDFInfo
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- CN103891711A CN103891711A CN201410115175.7A CN201410115175A CN103891711A CN 103891711 A CN103891711 A CN 103891711A CN 201410115175 A CN201410115175 A CN 201410115175A CN 103891711 A CN103891711 A CN 103891711A
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- seminal fluid
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- lateolabrax japonicus
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Abstract
The invention provides a treatment method for seminal fluid of lateolabrax japonicus. The method comprises following steps of (1) diluting and uniformly mixing the seminal fluid of the lateolabrax japonicus and an antifreeze fluid according to a volume ratio of 1:3 to obtain a diluted mixed liquor; (2) precooling the diluted mixed liquor for 3 minutes at 4 DEG C; (3) cooling down section by section; and (4) subpackaging and preserving. By adopting the treatment method for the seminal fluid of the lateolabrax japonicus, the problems of short time for producing sperm and a small amount of sperm of male lateolabrax japonicus parent fishes as well as non-unified maturity performance of signal female and male lateolabrax japonicus are solved; the difficulty of long distance transport of the seminal fluid of lateolabrax japonicus for long time is overcome; the preserved seminal fluid of the lateolabrax japonicus can provide materials for heterogeneous thelykaryon induction breeding any time and any where, and thus the method is beneficial to sustainable breeding of the lateolabrax japonicus, protection of the genetic diversity of the lateolabrax japonicus, and breeding of lateolabrax japonicus strains; the treatment method has great significance in breeding, genetic breeding and genetic resource protection of the lateolabrax japonicus.
Description
Technical field
The present invention relates to fish genetic technical field, relate in particular to a kind of processing method of spending perch seminal fluid.
Background technology
Flower perch belongs to Perciformes ， Sushi section, and perch belongs to flower.Be commonly called as: perch, jewfish, claim again a day reality perch.It is individual large, meat Jiamei, strong adaptability, growth is fast, feeding habits are wide, the nutrition contents such as protein, calcium, phosphorus, iron, vitamin, nicotinic acid, mcg vitamin B1 are abundant, are the carnivorous cultured fishes of the famous and precious seawater of China, are distributed widely in Bohai and Yellow Seas, the East Sea, Japan and marine site, the Korea peninsula are the coastal endemic species of the Pacific Northwest.Since the nineties in 20th century, through the development of nearly more than 20 years, China spent perch cultured output to reach more than 90,000 ton, ranks first of marine fish.But flower perch aquaculture is also faced with unprecedented challenge, and subject matter has: germplasm is degenerated, disease increases, aquaculture cost rising etc.The top-quality wild flowers perch of flower perch germplasm population quantity falls sharply, and parent population reproductive performance is constantly degenerated, the basic research weaknesses such as flower perch genetic breeding and breeding, and key technology does not obtain due attention, has had a strong impact on the sustainable development of colored perch aquaculture.
Present stage spends the natural breed of perch because the male and female individual generation gamete time is short, and the sexual maturity of male and female parent population is asynchronous, may cause being fertilized; The needed seminal fluid of artificial breeding adopts artificial belly extrusion to obtain, flower perch seminal fluid is leaving after spermary, owing to not taking any treatment measures, make the vigor of flower perch seminal fluid decline fast, can not preserve for a long time, cannot carry out long-distance transportation, be subject to the restriction of time and regional context, sperm quality can be along with for many generations accumulating decline, and excellent genes can be lost because of hybridization.
Summary of the invention
The object of the invention is to solve the defect that above-mentioned prior art exists, a kind of processing method of spending perch seminal fluid is provided.
A processing method of spending perch seminal fluid, comprises the following steps:
(1) by flower perch seminal fluid and anti frozen liquid by volume 1:3 dilution proportion mix;
(2) precooling treatment, by dilution after mixed liquor 4 DEG C of precoolings 3 minutes;
(3) after precooling, process with segmented mode cooling;
(4) packing is preserved;
Wherein, anti frozen liquid described in step 1 is made up of dilution, antifreeze and additive; Wherein, dilution is NaCl solution: 8.775g/L; The concentration of freezeproof protectant is 20%; Additive is bovine serum albumin(BSA): 10g/L, glucose: 10g/L, terramycin: 1g/L;
In step 3, described segmented mode cooling is processed and is comprised: first the mixed liquor after precooling treatment is stopped 5 minutes at the above 10cm of liquid nitrogen container liquid level, then stop 5 minutes at the above 4cm of liquid nitrogen container liquid level, finally in the above 1cm stop of liquid nitrogen container liquid level was directly absorbed in liquid nitrogen after 3 minutes, preserve for a long time.
Further, the processing method of colored perch seminal fluid as above, after step 4, also comprise the step 5 of thawing, being about to process through liquid nitrogen colored perch that packing preserves freezes essence and directly puts into 37 DEG C of water-baths and thaw 50-60 second, constantly shake flower perch is frozen essence during this time, is then placed in room temperature to melting completely, then in nature seawater, activates.
Further, the processing method of colored perch seminal fluid as above, described activation comprises inserts the essence of freezing after thawing through scalding and is added with in the nature seawater of 5% bovine serum albumin(BSA) and activate.
Further, the processing method of described colored perch seminal fluid, before step 1, also comprises obtaining of colored perch seminal fluid as mentioned above, comprises the following steps:
(1) choose 3-4 age, the male flower perch parent population of body weight 3-4kg is anaesthetized;
(2) press the amount intramuscular injection HCG of 1000U/kg;
After (3) three days, adopt belly extrusion to obtain seminal fluid, the more than 95% colored perch seminal fluid of the vigor of getting.
Further, the processing method of described colored perch seminal fluid as mentioned above, described freezeproof protectant is methyl-sulfoxide.
Further, the as mentioned above processing method of described colored perch seminal fluid, after 1:3 dilution proportion mixes by volume by flower perch seminal fluid and anti frozen liquid in step 1, is packaged in cryopreservation tube the cryopreservation tube that described cryopreservation tube is 2ml.
Further, the processing method of described colored perch seminal fluid as mentioned above, described segmented mode cooling is processed and is comprised employing programmed cooling instrument programmed cooling.
The processing method of colored perch seminal fluid provided by the invention, by breeding period its semen cryopreservation being got up flower perch, the rewarming that thaws whenever and wherever possible obtains the higher seminal fluid of vigor, solved male flower perch parent population produce the essence time short, produce the few difficulty of essence amount, solve the ripe nonsynchronous problem of colored perch male and female individuality, solved colored perch sperm and cannot grow distance, long-time difficulty of transporting; The colored perch sperm of preserving, can be whenever and wherever possible for allos thelykaryon induced breeding provides material, is conducive to the sustainable cultivation of flower perch, is conducive to the protection of flower perch genetic diversity, is conducive to the seed selection of flower perch strain; In preserving, breeding, genetic breeding and the germ plasm resource of flower perch has great importance.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below technical scheme in the present invention be clearly and completely described, obviously, described embodiment is the present invention's part embodiment, instead of whole embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment:
The processing method of colored perch seminal fluid provided by the invention, comprises the following steps:
(1) gather the 3-4 age in breeding period, body weight 3-4kg, the colored perch milter that gonad development is good, milter is pulled from culturing pool out to rear anesthesia, press 1000U/kg intramuscular injection HCG, three days afterwards with distilled water flushing gonopore, clean, push gently belly and obtain fresh semen, then by vigor higher than 95% seminal fluid and anti frozen liquid by volume 1:3 dilution proportion after mixing, join in the cryopreservation tube of 2ml; Described anti frozen liquid is made up of dilution, antifreeze and additive; Wherein, dilution is NaCl solution: 8.775g/L; Freezeproof protectant (dimethyl sulfoxide (DMSO)) is the concentration of 20%(freezeproof protectant, V/V) DMSO(dimethyl sulfoxide (DMSO)); Additive is, bovine serum albumin(BSA): 10g/L, glucose: 10g/L, terramycin: 1g/L.
(2) cryopreservation tube is placed in to 4 DEG C of refrigerator precoolings 3 minutes, then put into micro metal tank with the processing of segmentation cooling method, first stop 5 minutes at the above 10cm of liquid nitrogen container liquid level, then stop 5 minutes at the above 4cm of liquid nitrogen container liquid level, finally after the above 1cm of liquid nitrogen container liquid level stops 3 minutes, directly put into liquid nitrogen (196 DEG C), packing is preserved;
(3) the colored perch of processing packing preservation through liquid nitrogen is frozen to the row that progresses greatly and thaw, it is directly put into 37 DEG C of water-baths and thaw 50-60 second, constantly shake during this time cryopreservation tube, be then placed in room temperature to melting completely;
(4), by the line activating that progresses greatly that freezes after thawing, described active mode is: first on slide, drip through scalding and be added with 5%BSA nature seawater, then adding 1-2 μ L to freeze essence with liquid-transfering gun, suction is beaten and made for 3-5 time it fully mix.
The essence of freezing after activating on slide, is got immediately 3 visuals field by the present embodiment, adopt area of computer aided sperm analysis system counting statistics rate of motion, after thawing after testing, Sperm motility is 90.87 ± 2.25%, with not remarkable (P < 0.05) of fresh smart difference.
Beneficial effect of the present invention:
1. the present invention adopts 20%(V/V) methyl-sulfoxide is freezeproof protectant, keeps the better infiltrative while to reduce again toxicity;
2. in anti frozen liquid of the present invention, added bovine serum albumin(BSA), the plasma membrane of sperm has been played a very good protection;
3. in anti frozen liquid of the present invention, added appropriate glucose, its impermeability protective effect has improved the freezing tolerance of colored perch sperm;
The present invention before lowering the temperature fresh essence and anti frozen liquid mix, in 4 DEG C of refrigerators, precooling is directly put into liquid nitrogen container and is directly carried out segmentation cooling after 3 minutes, has saved the time, easy to operation;
5. when segmentation of the present invention is lowered the temperature, adopt with the above 10cm precooling of liquid nitrogen surface, make frozen sperm degree of safety cross " dangerous temperature district ", then the rate of temperature fall of the above 4cm of liquid nitrogen surface enters stable state fast, last liquid nitrogen surface directly immerses in liquid nitrogen after stopping above the short period, the whole temperature-fall period time is short, practical;
6. after frozen sperm of the present invention takes out from liquid nitrogen, adopt two step freezing processes, 37 DEG C and 20 DEG C of two step are thawed, and can make sperm fast speed cross annealed zone, and the while has been avoided again the too high damage causing of local temperature;
7. cooling system of the present invention only needs a liquid nitrogen container, and equipment is simple, easy operating, freeze essence thaw after anabiosis rate high, after activating, rate of motion is higher than 90%, active basic identical with fresh essence.
8. the present invention has added the terramycin of 1g/L in anti frozen liquid, and the harmful microorganism in anti frozen liquid is played to obvious inhibitory action;
The present invention lower the temperature before flower perch seminal fluid and anti frozen liquid mix, 4 DEG C of balances 3 minutes, can ensure that freezeproof protectant fully penetrates into colored perch sperm inside, can not cause excessive infringement to embryo because of the toxic action of freezeproof protectant itself again;
10. the present invention adopts the sea-water activated colored perch seminal fluid of sterilization that adds 5%BSA, and after effectively preventing from thawing, flower perch spermatic flagellum is adhered mutually.
Finally it should be noted that: above embodiment only, in order to technical scheme of the present invention to be described, is not intended to limit; Although the present invention is had been described in detail with reference to previous embodiment, those of ordinary skill in the art is to be understood that: its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement; And these amendments or replacement do not make the essence of appropriate technical solution depart from the spirit and scope of various embodiments of the present invention technical scheme.
Claims (7)
1. a processing method of spending perch seminal fluid, is characterized in that: comprise the following steps:
(1) by flower perch seminal fluid and anti frozen liquid by volume 1:3 dilution proportion mix;
(2) precooling treatment, by dilution after mixed liquor 4 DEG C of precoolings 3 minutes;
(3) after precooling, process with segmented mode cooling;
(4) packing is preserved;
Wherein, anti frozen liquid described in step 1 is made up of dilution, antifreeze and additive; Wherein, dilution is NaCl solution: 8.775g/L; The concentration of freezeproof protectant is 20%; Additive is bovine serum albumin(BSA): 10g/L, glucose: 10g/L, terramycin: 1g/L;
In step 3, described segmented mode cooling is processed and is comprised: first the mixed liquor after precooling treatment is stopped 5 minutes at the above 10cm of liquid nitrogen container liquid level, then stop 5 minutes at the above 4cm of liquid nitrogen container liquid level, finally in the above 1cm stop of liquid nitrogen container liquid level was directly absorbed in liquid nitrogen after 3 minutes, preserve for a long time.
2. the processing method of colored perch seminal fluid according to claim 1, it is characterized in that: after step 4, also comprise the step 5 of thawing, being about to process through liquid nitrogen colored perch that packing preserves freezes essence and directly puts into 37 DEG C of water-baths and thaw 50-60 second, constantly shake flower perch is frozen essence during this time, then be placed in room temperature to melting completely, then in nature seawater, activate.
3. the processing method of colored perch seminal fluid according to claim 2, is characterized in that: described activation comprises inserts the essence of freezing after thawing through scalding and be added with in the nature seawater of 5% bovine serum albumin(BSA) and activate.
4. the processing method of colored perch seminal fluid according to claim 1, is characterized in that: before step 1, also comprise obtaining of colored perch seminal fluid, comprise the following steps:
(1) choose 3-4 age, the male flower perch parent population of body weight 3-4kg is anaesthetized;
(2) press the amount intramuscular injection HCG of 1000U/kg;
After (3) three days, adopt belly extrusion to obtain seminal fluid, the more than 95% colored perch seminal fluid of the vigor of getting.
5. according to the processing method of the arbitrary described colored perch seminal fluid of claim 1-5, it is characterized in that: described freezeproof protectant is methyl-sulfoxide.
6. according to the processing method of the arbitrary described colored perch seminal fluid of claim 1-5, it is characterized in that: after 1:3 dilution proportion mixes by volume by flower perch seminal fluid and anti frozen liquid in step 1, be packaged in cryopreservation tube the cryopreservation tube that described cryopreservation tube is 2ml.
7. according to the processing method of the arbitrary described colored perch seminal fluid of claim 1-5, it is characterized in that: described segmented mode cooling is processed and comprised employing programmed cooling instrument programmed cooling.
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| CN201410115175.7A CN103891711B (en) | 2014-03-26 | 2014-03-26 | The processing method of flower perch seminal fluid |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062954A (en) * | 2015-08-17 | 2015-11-18 | 宁波市海洋与渔业研究院 | Ultra-low temperature frozen mackerel semen thawing method |
| CN110226592A (en) * | 2019-06-29 | 2019-09-13 | 中国海洋大学 | A kind of flat Rockfish sperm cryopreservation method of Xu Shi |
| CN112293411A (en) * | 2020-11-27 | 2021-02-02 | 大连海洋大学 | Low-temperature preservation liquid and preservation method for sperms of echinococcus intermedius |
| CN110036950B (en) * | 2019-04-23 | 2021-07-30 | 青岛农业大学 | Artificial crossbreeding method for mariculture of rainbow trout |
| CN115349496A (en) * | 2022-08-19 | 2022-11-18 | 湖南师范大学 | Method for inducing gynogenesis of micropterus salmoides by siniperca chuatsi sperms |
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| CN1596670A (en) * | 2004-07-27 | 2005-03-23 | 中国水产科学研究院黄海水产研究所 | Practicalization method for frozen preserving sperm of fish |
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| CN1596670A (en) * | 2004-07-27 | 2005-03-23 | 中国水产科学研究院黄海水产研究所 | Practicalization method for frozen preserving sperm of fish |
Non-Patent Citations (2)
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062954A (en) * | 2015-08-17 | 2015-11-18 | 宁波市海洋与渔业研究院 | Ultra-low temperature frozen mackerel semen thawing method |
| CN110036950B (en) * | 2019-04-23 | 2021-07-30 | 青岛农业大学 | Artificial crossbreeding method for mariculture of rainbow trout |
| CN110226592A (en) * | 2019-06-29 | 2019-09-13 | 中国海洋大学 | A kind of flat Rockfish sperm cryopreservation method of Xu Shi |
| CN112293411A (en) * | 2020-11-27 | 2021-02-02 | 大连海洋大学 | Low-temperature preservation liquid and preservation method for sperms of echinococcus intermedius |
| CN115349496A (en) * | 2022-08-19 | 2022-11-18 | 湖南师范大学 | Method for inducing gynogenesis of micropterus salmoides by siniperca chuatsi sperms |
| CN115349496B (en) * | 2022-08-19 | 2024-01-16 | 湖南师范大学 | Method for inducing gynogenesis of micropterus salmoides by siniperca chuatsi sperms |
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| CN103891711B (en) | 2015-10-21 |
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