CN103891711B - The processing method of flower perch seminal fluid - Google Patents
The processing method of flower perch seminal fluid Download PDFInfo
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- CN103891711B CN103891711B CN201410115175.7A CN201410115175A CN103891711B CN 103891711 B CN103891711 B CN 103891711B CN 201410115175 A CN201410115175 A CN 201410115175A CN 103891711 B CN103891711 B CN 103891711B
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Abstract
The invention provides a kind of processing method spending perch seminal fluid, the method comprises the following steps: (1) will spend perch seminal fluid and anti frozen liquid by volume 1:3 dilution proportion mix; (2) precooling treatment, by the mixed liquor after dilution 4 DEG C of precoolings 3 minutes; (3) process with segmented mode cooling after precooling; (4) packing is preserved.The processing method of colored perch seminal fluid provided by the invention, solves male flower perch parent population and produces the difficulty that the essence time is short, product essence amount is few, solves the ripe nonsynchronous problem of colored perch male and female individuality, solves the difficulty that colored perch sperm cannot grow distance, transport for a long time; The colored perch sperm preserved, whenever and wherever possible for allos thelykaryon induced breeding provides material, can be conducive to the sustainable cultivation of flower perch, be conducive to the protection of flower perch genetic diversity, be conducive to the seed selection of flower perch strain; Have great importance in the flower breeding of perch, genetic breeding and Germ-plasma resources protection.
Description
Technical field
The present invention relates to fish genetic technical field, particularly relate to a kind of processing method spending perch seminal fluid.
Background technology
Flower perch belongs to Perciformes ， Sushi section, and perch belongs to flower.Be commonly called as: perch, jewfish, also known as 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 enrich, and are the famous and precious carnivorous cultured fishes of 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 spends 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: plant that matter is degenerated, disease increases, aquaculture cost rising etc.Flower perch kind matter top-quality wild flowers perch population quantity falls sharply, and parent population reproductive performance is constantly degenerated, and basic research such as flower perch genetic breeding and breeding etc. is weak, and key technology does not obtain due attention, has had a strong impact on the sustainable development of colored perch aquaculture.
Present stage spend the natural breed of perch due to male and female individuality produce the gamete time short, the sexual maturity of male and female parent population is asynchronous, may cause being fertilized; Seminal fluid required for artificial breeding adopts artificial belly extrusion to obtain, flower perch seminal fluid is after leaving spermary, owing to not taking any treatment measures, the vigor of flower perch seminal fluid is made to decline fast, can not preserve for a long time, cannot long-distance transportation be carried out, by the restriction of Time and place region, sperm quality can 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 the defect solving the existence of above-mentioned prior art, a kind of processing method spending perch seminal fluid is provided.
Spend a processing method for perch seminal fluid, comprise the following steps:
(1) will spend perch seminal fluid and anti frozen liquid by volume 1:3 dilution proportion mix;
(2) precooling treatment, by the mixed liquor after dilution 4 DEG C of precoolings 3 minutes;
(3) process with segmented mode cooling after precooling;
(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 process comprises: 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, be finally directly absorbed in liquid nitrogen after the above 1cm of liquid nitrogen container liquid level stops 3 minutes and preserve for a long time.
Further, the processing method of colored perch seminal fluid as above, after step 4, also comprise step 5 of thawing, the colored perch being about to preserve through liquid nitrogen process packing is frozen essence and directly puts into 37 DEG C of water-baths and thaw 50-60 second, period constantly shakes colored perch and freezes essence, being then placed in room temperature to melting completely, then activating in nature seawater.
Further, the processing method of flower perch seminal fluid as above, described activation comprises inserts through scalding and is added with in the nature seawater of 5% bovine serum albumin(BSA) activate the essence of freezing after thawing.
Further, the processing method of colored perch seminal fluid described as mentioned above, before step 1, also comprises the acquisition of colored perch seminal fluid, comprises the following steps:
(1) choose 3-4 age, the male flower perch parent population of body weight 3-4kg is anaesthetized;
(2) by the amount intramuscular injection HCG of 1000U/kg;
Within (3) three days, adopt belly extrusion to obtain seminal fluid afterwards, get the colored perch seminal fluid of vigor more than 95%.
Further, the processing method of colored perch seminal fluid described as mentioned above, described freezeproof protectant is methyl-sulfoxide.
Further, the processing method of as mentioned above described colored perch seminal fluid, after spending perch seminal fluid and anti frozen liquid in step 1 1:3 dilution proportion mixing by volume, be packaged in cryopreservation tube, described cryopreservation tube will be the cryopreservation tube of 2ml.
Further, the processing method of colored perch seminal fluid described as mentioned above, described segmented mode cooling process comprises 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, solve male flower perch parent population and produce the difficulty that the essence time is short, product essence amount is few, solve the ripe nonsynchronous problem of colored perch male and female individuality, solve the difficulty that colored perch sperm cannot grow distance, long-time transport; The colored perch sperm preserved, whenever and wherever possible for allos thelykaryon induced breeding provides material, can be conducive to the sustainable cultivation of flower perch, be conducive to the protection of flower perch genetic diversity, be conducive to the seed selection of flower perch strain; Have great importance in the flower breeding of perch, genetic breeding and Germ-plasma resources protection.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, 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 embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained 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) the 3-4 age being in breeding period is gathered, the colored perch milter that body weight 3-4kg, gonad development are good, milter is pulled out rear anesthesia from culturing pool, by 1000U/kg intramuscular injection HCG, three days afterwards with distilled water flushing gonopore, clean, gently extrude belly obtain fresh semen, then by vigor higher than 95% seminal fluid and anti frozen liquid by volume 1:3 dilution proportion join in the cryopreservation tube of 2ml after mixing; Described anti frozen liquid is made up of dilution, antifreeze and additive; Wherein, dilution is NaCl solution: 8.775g/L; The concentration that freezeproof protectant (dimethyl sulfoxide (DMSO)) is 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 4 DEG C of refrigerator precoolings 3 minutes, then put into micro metal tank with the process of segmentation cooling method, namely first 5 minutes are stopped at the above 10cm of liquid nitrogen container liquid level, then 5 minutes are stopped 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 preserved through liquid nitrogen process packing is frozen the row that progresses greatly to thaw, it is directly put into 37 DEG C of water-baths and thaw 50-60 second, period constantly shakes cryopreservation tube, is then placed in room temperature to melting completely;
(4), will freeze the line activating that progresses greatly after thawing, described active mode is: first drip on slide through scalding and be added with 5%BSA nature seawater, then adding 1-2 μ L with liquid-transfering gun and freeze essence, and suction is beaten and made for 3-5 time it fully mix.
Essence of freezing after activation is got 3 visuals field immediately by the present embodiment on slide, adopt computer aided pass design counting statistics rate of motion, after thawing after testing, Sperm motility is 90.87 ± 2.25%, with fresh smart difference remarkable (P < 0.05).
Beneficial effect of the present invention:
1. the present invention adopts 20%(V/V) methyl-sulfoxide is freezeproof protectant, keeps better infiltrative and again reduces toxicity simultaneously;
2. add bovine serum albumin(BSA) in anti frozen liquid of the present invention, the plasma membrane of sperm is played a very good protection;
3. add appropriate glucose in anti frozen liquid of the present invention, its impermeability protective effect improves the freezing tolerance of colored perch sperm;
4. 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, saves the time, easy to operation;
5. during segmentation cooling of the present invention, adopt with the above 10cm precooling of liquid nitrogen surface, frozen sperm is spent safely in " dangerous temperature district ", then the rate of temperature fall of the above 4cm of liquid nitrogen surface enters stable state fast, directly immerse in liquid nitrogen after more than last liquid nitrogen surface stopping 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 sperm fast speed can be made to cross annealed zone, turn avoid the too high damage caused of local temperature simultaneously;
7. cooling system of the present invention only needs a liquid nitrogen container, and equipment is simple, is easy to operation, freeze essence thaw after anabiosis rate high, after activating, rate of motion is higher than 90%, substantially identical with fresh smart activity.
8. the present invention adds the terramycin of 1g/L in anti frozen liquid, plays obvious inhibitory action to the harmful microorganism in anti frozen liquid;
9. the present invention spends perch seminal fluid and anti frozen liquid to mix before lowering the temperature, and 4 DEG C balance 3 minutes, can ensure that freezeproof protectant fully penetrates into colored perch sperm inner, can not cause excessive infringement again because of the toxic action of freezeproof protectant itself to embryo;
10. the present invention adopts and adds the sea-water activated colored perch seminal fluid of sterilization of 5%BSA, and after effectively preventing from thawing, flower perch spermatic flagellum is adhered mutually.
Last it is noted that above embodiment is only in order to illustrate technical scheme of the present invention, be not intended to limit; Although with reference to previous embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature; 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 (4)
1. spend a processing method for perch seminal fluid, it is characterized in that: comprise the following steps:
(1) will spend perch seminal fluid and anti frozen liquid by volume 1:3 dilution proportion mix;
(2) precooling treatment, by the mixed liquor after dilution 4 DEG C of precoolings 3 minutes;
(3) process with segmented mode cooling after precooling;
(4) packing is preserved;
Wherein, described in step (1), anti frozen liquid is made up of dilution, freezeproof protectant 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 process comprises: 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, be finally directly absorbed in liquid nitrogen after the above 1cm of liquid nitrogen container liquid level stops 3 minutes and preserve for a long time;
After step (4), also comprise step of thawing (5), the colored perch being about to preserve through liquid nitrogen process packing is frozen essence and directly puts into 37 DEG C of water-baths and thaw 50-60 second, and period constantly shakes colored perch and freezes essence, then being placed in room temperature to melting completely, then activating in nature seawater;
Described activation comprises inserts through scalding and is added with in the nature seawater of 5% bovine serum albumin(BSA) activate the essence of freezing after thawing.
2. the processing method of colored perch seminal fluid according to claim 1, is characterized in that: described freezeproof protectant is methyl-sulfoxide.
3. the processing method of colored perch seminal fluid according to claim 1, is characterized in that: after spending perch seminal fluid and anti frozen liquid in step (1) 1:3 dilution proportion mixing by volume, be packaged in cryopreservation tube, described cryopreservation tube will be the cryopreservation tube of 2ml.
4. the processing method of colored perch seminal fluid according to claim 1, is characterized in that: described segmented mode cooling process comprises 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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| 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 |
| CN112293411B (en) * | 2020-11-27 | 2021-09-28 | 大连海洋大学 | Low-temperature preservation liquid and preservation method for sperms of echinococcus intermedius |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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)
| Title |
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| 花鲈精子生理特性及其精液超低温冷冻保存;洪万树等;《海洋学报》;19960313;第18卷(第2期);第97-104页 * |
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