CN102630664B - Anticoagulation preserving fluid for fish sperms, and preparation method and application thereof - Google Patents
Anticoagulation preserving fluid for fish sperms, and preparation method and application thereof Download PDFInfo
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- CN102630664B CN102630664B CN 201210081749 CN201210081749A CN102630664B CN 102630664 B CN102630664 B CN 102630664B CN 201210081749 CN201210081749 CN 201210081749 CN 201210081749 A CN201210081749 A CN 201210081749A CN 102630664 B CN102630664 B CN 102630664B
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Abstract
The invention discloses anticoagulation preserving fluid for fish sperms, and a preparation method and application thereof. The preparation method includes: firstly, acquiring human seminal plasma; secondly, clarifying the human seminal plasma; and thirdly, preparing the anticoagulation preserving fluid for fish sperm. The anticoagulation preserving fluid for fish sperms prepared by the method not only is capable of effectively resisting sperm damage and sperm fertility influence caused by coagulation of the fish sperms, but also can be used for storing information carried by each sperm to the maximum extent and guaranteeing maximum utilization of the thawed sperms. The anticoagulation preserving fluid for fish sperms has high practical significance and application value for low-temperature freeze storage of rare fish germplasm resources.
Description
Technical field
The invention belongs to the cryobiology research field, be specifically related to a kind of fish sperm anti-freezing collection and preserve liquid and its preparation method and application.
Background technology
Sperm agglutination is a kind of phenomenon more common in the sperm super-low temperature freezing process, slight aggegation can make the part spermatogenesis crosslinked, the motion of restriction sperm, serious aggegation impel a large amount of spermatogenesis films to merge and in the form of sheets, strip, lumps, and single sperm is lost.The inventor Dong Qiao of present patent application is fragrant wait once description detailed in the research frozen to the Pacific oyster sperm super-low temperature this phenomenon (see for details: Qiaoxiang Dong, Changjiang Huang, et al. Control of sperm concentration is necessary for standardization of sperm cryopreservation in aquatic species:Evidence from sperm agglutination in oysters. Cryobiology, 2007,54:87-98).
Dong Qiaoxiang etc. find when how to avoid superfreeze process Mesichthyes spermatogenesis coagulation problems in research, and the zebra fish sperm can discharge the composition of certain short sperm cohesion in the superfreeze process.Process without freezing zebra fish sperm because freeze smart supernatant with the zebra fish of collecting, can make equally the zebra fish sperm coacervation occur.On this basis, they find to freeze with zebra fish the cohesion that smart supernatant can be induced other fish (Puntius tetrazona, lamp) end to end sperm equally, but to human sperm's short solidifying effect of tool not.For this reason, they think that Human seminal plasma may contain the poly-composition of certain anti-freezing.Therefore, they process the zebra fish sperm with Human seminal plasma, thereby have found the effect of this anti-fish sperm cohesion of Human seminal plasma.They are according to being engaged in for many years experience that the fish cryogenic freezing preserves and to the variation of film fat content in sperm freezing, think that this characteristic of congealing of fish sperm may stem from sphingomyelins in the sperm after birth.They when processing the zebra fish sperm with the sphingomyelins liposome, found the identical coacervation that produces with the cryogenic freezing process, with the liposome of other lipid preparation without similarly acting on.Therefore, tentative confirmation be that sphingomyelins induces fish sperm that coacervation occurs in refrigerating process, and Human seminal plasma can effectively resist this fish sperm cohesion of being induced by sphingomyelins.
Effectively resist the method for condensing in the fish sperm refrigerating process by the processing with Human seminal plasma.Also there is not yet at present relevant report both at home and abroad.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of fish sperm anti-freezing collection to preserve liquid and its preparation method and application, condense in the superfreeze process effectively to prevent fish sperm, simple and be applicable to other a lot of fish.
For achieving the above object, the present invention adopts following technical scheme:
A kind of fish sperm anti-freezing collection is preserved the preparation method of liquid, comprises the following steps:
1) acquisition of Human seminal plasma;
2) purifying of Human seminal plasma;
3) fish sperm anti-freezing collection is preserved the preparation of liquid.
Further, above-mentioned steps 1) comprising: obtain fresh semen from healthy human body, preserve in 35-40 ℃ of incubator and treat its liquefaction; The seminal fluid that liquefies is divided be filled to 1.5ml centrifuge tube (1ml/ pipe), then high speed centrifugation (10000 rev/mins centrifugal 10 minutes), draw the upper strata refining (the refining volume of absorption be centrifugal use the seminal fluid volume 1/2 to 2/3, avoid being drawn to precipitation) to the 1.5ml centrifuge tube, save backup.
Further, above-mentioned steps 2) comprising: with above-mentioned steps 1) in-16 to-20 ℃ of refrigerator and cooled of refining of obtaining freeze, take out after the refining freeze all, naturally thaw under room temperature, high speed centrifugation (10000 rev/mins centrifugal 10 minutes) is removed the impurity of separating out, and by above-mentioned condition again repeated freezing, thaw, once centrifugal, obtain impure refining still less; At last, with 0.22-0.45 μ m membrane filtration, the refining that obtains purifying saves backup in 0-4 ℃.
further, above-mentioned steps 3) comprising: first the refining after purifying is mixed with the volume ratio of 1:1 to 1:3 with the Hank's balance solution, then use osmometer to the osmotic pressure of this mixed solution detect (preparation complete after or detect when using all can), with near the osmotic pressure of this kind fish refining or be beneficial to freeze proof osmotic pressure and be advisable (when needing to add some impermeability antifreeze, consider that osmotic pressure that antifreeze brings changes to avoid activation or the damage of sperm), and use ultra-pure water (reduction), or glucose (lifting) is regulated osmotic pressure, and (osmotic pressure of fresh-water fishes spermatozoa preservative fluid should be between 260-310mOs/kg to target osmotic pressure, the osmotic pressure of fish spermatozoa preservative fluid should be between 150-200mOs/kg).Only needing to preserve liquid suspension sperm with this during concrete the use gets final product.
The present invention also provides the fish sperm anti-freezing collection of said method preparation to preserve liquid, take Human seminal plasma as raw material, mixes making after purifying with the Hank's balance solution with the volume ratio of 1:1 to 1:3.
The present invention further provides above-mentioned fish sperm anti-freezing collection and preserve the application of liquid in preventing the fish sperm cohesion.
The beneficial effect that the present invention produces is as follows:
The prepared fish sperm anti-freezing collection of the inventive method is preserved liquid, can effectively avoid by the caused Sperm lesion of sperm cohesion, preserve to greatest extent the entrained information of each sperm, minimizing is due to the impact of the caused sperm fertilizing ability of sperm cohesion, reduce the interference that sperm condenses the Sperm Parameters analysis that brings, thereby make each frozen sperm of experience can be utilized by amplitude peak ground after thawing.
Description of drawings
Fig. 1 shows the Technology Roadmap that the invention provides for anti-fish sperm condensing method;
Fig. 2 shows the design sketch (A: control group, B: the Human seminal plasma processed group) of the anti-zebra fish freeze agglomeration of Human seminal plasma;
Fig. 3 shows the sperm flocculating result that the liposome-induced sperm of sphingomyelins condenses and the anti-sphingomyelins of Human seminal plasma causes (A: the zebra fish sperm figure before the sphingomyelins test fluid is processed; Performance figure after the control group that the B:Hank's balance solution suspends is processed through the sphingomyelins test fluid; C: the as a result figure of Human seminal plasma processed group after the sphingomyelins test fluid is processed; A ': the zebra fish sperm amplification effect figure before the sphingomyelins test fluid is processed; B ': the performance amplification effect figure after the control group that the Hank's balance solution suspends is processed through the sphingomyelins test fluid; C ': the as a result enlarged drawing of Human seminal plasma processed group after the sphingomyelins test fluid is processed).
Embodiment
Be noted that following illustrating is all exemplary, be intended to the invention provides further invention.Except as otherwise noted, all Science and Technology terms used herein have the identical meanings of usually understanding with the technical field of the invention personnel.
Be described further below in conjunction with accompanying drawing:
A kind of fish sperm anti-freezing collection is preserved preparation method's (seeing Fig. 1) of liquid, and concrete implementation step is as follows:
One, the acquisition of Human seminal plasma
(1) seminal fluid obtains: select healthy volunteer (without venereal disease, blood born diseases, relevant cause of disease detects negative).Obtain fresh semen by the masturbation method, preserve in 37 ℃ of incubators until its liquefaction (after 30 minutes the seminal fluid of liquefaction discard need not) not yet.
(2) separate refining: the seminal fluid of liquefaction is divided be filled to 1.5ml centrifuge tube (1ml/ pipe) with 4 ℃, 10000 rev/mins centrifugal 10 minutes, absorption upper strata refining standby (the refining volume of absorption be centrifugal use the seminal fluid volume 1/2, avoid being drawn to precipitation).
Two, the purifying of Human seminal plasma
(1) refining freeze thawing treatment: the refining of obtaining is frozen in-20 ℃ of refrigerator and cooled, take out after the refining freeze all, naturally thaw under room temperature and (note: when freezing, need to use the packing of 1.5ml centrifuge tube; All need keep centrifuge tube upright in freezing and course of defrosting, and it is molten again to avoid rocking the impurity that centrifuge tube separated out to avoid in frozen-thaw process after thawing).
(2) centrifugal: the refining after thawing (be placed in former centrifuge tube in) is with 4 ℃, removes the impurity (noting: repeat refining freeze thawing, centrifugal two steps, can obtain impure refining still less) of separating out in centrifugal 10 minutes for 10000 rev/mins.
(3) filter: the refining after centrifugal is filtered with 0.45 μ m polycarbonate leaching film, and the refining that obtains purifying saves backup in-20 ℃.
Three, fish sperm anti-freezing collection is preserved the preparation of liquid
(1) refining dilution: the refining after purifying is mixed with the Hank's balance solution with the volume ratio of 1:2.
(2) adjust osmotic pressure: use osmometer (the German GONOTEC OSMOMAT-30-M of company type) that the osmotic pressure of this mixed solution is detected; with near the osmotic pressure of this kind fish refining or be beneficial to freeze proof osmotic pressure and be advisable (when needing to add some impermeability antifreeze; consider that osmotic pressure that antifreeze brings changes to avoid activation or the damage of sperm); by adding ultra-pure water (reduction), or the osmotic pressure that glucose (lifting) resists aggegation protection liquid finely tunes to be adjusted to OK range.(solution osmotic pressure that be used for to preserve the fresh-water fishes sperm should be 260-310mOs/kg, if seawater fish osmotic pressure should be 150-200mOs/kg, fresh water oviparity fish generally can need not to adjust, and adjust when finding that this preservation liquid can activate fish sperm again).Can reach the poly-purpose of anti-freezing with the required frozen fish sperm of this liquid suspension.
Below in conjunction with specific embodiment, the present invention is further detailed.
Example 1, be applied to freezing caused sperm cohesion
Usually, should avoid occurring the sperm cohesion as far as possible in the sperm freezing process, thereby reach frozen effect preferably.Therefore, obvious cohesion occurred after sperm freezing, thought that selected freezing scheme is not the frozen of very suitable this kind sperm.The most extreme situation is exactly not add any antifreeze direct plunge into Liquid Nitrogen, can cause serious sperm cohesion.And Human seminal plasma can be significantly in the situation that this extreme its anticoagulation of performance, its concrete implementation step is as follows:
1) acquisition of Human seminal plasma:
Method is the same.
2) purifying of Human seminal plasma:
Method is the same.
3) preparation of liquid is preserved in the anti-freezing of zebra fish sperm:
Method is the same.
4) zebra fish sperm freezing:
Preserve to blow and beat in liquid in anti-freezing after the zebra fish spermary is taken out and discharge out sperm (with the Hank's balance solution in contrast), adjust sperm concentration to 10
8Individual/ml is distributed into straw (or cryopreservation tube), and then direct plunge into Liquid Nitrogen, take out after the sperm suspension fully charge and thaw, and observes in microscopically.The results are shown in Figure 3, Fig. 3 show thaw with Hank's balance solution zebra fish sperm in contrast after, seriously cohesion appears, keep that independently sperm concentration is extremely low, and use anti-freezing to preserve the processed group sperm high degree of dispersion of liquid, be evenly distributed, without coacervation.Illustrate that this method can perform well in freezing caused sperm cohesion.
Example 2, be applied to the sperm cohesion that sphingomyelins induces
Inventor's tentative confirmation sphingomyelins can induce fish sperm to condense in refrigerating process, thereby add sphingomyelins under non-freezing state in fish sperm, can cause that equally fish sperm condenses, but Human seminal plasma can resist the sphingomyelins liposome this blood coagulation enhancing effect we adopt different fingerlings to test, the results are shown in Table 1.
Table 1: different fish sperms add after Human seminal plasmas the anti-freezing effect and for the sphingomyelins sensitivity tests.
Annotate: except zebra fish, all the other all fishes of using are market and buy.
For describing this application in detail, the below is take zebra fish as example, and its concrete implementation step is as follows:
1) preparation of sphingomyelins liposome solutions: take 5mg sphingomyelins (sigma company) and be dissolved in 50 μ l methyl alcohol (domestic analysis is pure), add wherein Hank's balance solution 5ml by heating water bath to 50 ℃ with the liquid feeding rifle rapidly.4 ℃ save backup (effective in January).
2) preparation of sphingomyelins working solution: get 100 μ l sphingomyelins liposome solutions, add 400 μ l ultra-pure waters (the corresponding freshwater fish of ultra-pure water, if seawater fish adds the seawater of same volume or with the NaCl solution of isosmoticity).Working solution needs interim preparation.
3) acquisition of Human seminal plasma:
Method is the same.
4) preparation of liquid is preserved in the anti-freezing of zebra fish sperm:
Method is the same.
5) the sperm cohesion that causes of anti-sphingomyelins:
Preserve to blow and beat in liquid in anti-freezing after the zebra fish spermary is taken out and discharge out sperm (with the Hank's balance solution in contrast), adjust sperm concentration to 10 in sperm suspension
8Then individual/ml drips 20 μ l test fluid (the method preparation by introducing before wherein contains the sphingomyelins liposome), then adds 5 μ l sperm suspensions on slide, observe under dark-field microscope.The results are shown in Figure 3, can see that the processed group that adds the sphingomyelins liposome is with respect to control group, cohesion has appearred, and the processed group of using anti-freezing liquid is with respect to the processed group that has only added the sphingomyelins liposome, the sperm high degree of dispersion, be evenly distributed, this shows that this method can well be used for the caused zebra fish sperm cohesion of sphingomyelins.
The above is only the preferred embodiments of the present invention, should be understood that; for the those of ordinary skill in present technique; under the prerequisite that does not break away from core technology feature of the present invention, can also make some improvements and modifications, these retouchings and improvement also should belong to scope of patent protection of the present invention.
Claims (4)
1. the preparation method that fish sperm anti-freezing collection is preserved liquid, is characterized in that, comprises the following steps:
1) acquisition of Human seminal plasma: obtain fresh semen from healthy human body, preserve in the incubator of 35-40 ℃ and treat its liquefaction; The seminal fluid of liquefaction is divided and is filled to the 1.5ml centrifuge tube, 10000 rev/mins centrifugal 10 minutes, draw the upper strata refining to the 1.5ml centrifuge tube, save backup; The volume of wherein said upper strata refining be centrifugal use the seminal fluid volume 1/2 to 2/3;
2) Human seminal plasma that obtains the purifying of Human seminal plasma: with step 1) freezes in-16 ℃ to-20 ℃ refrigerator and cooled, take out after the refining freeze all, naturally thaw under room temperature, 10000 rev/mins of centrifugal impurity of separating out with removal in 10 minutes, and by above-mentioned condition again repeated freezing, thaw, once centrifugal, obtain impure refining still less; At last, filter with 0.22-0.45 μ m polycarbonate leaching film, the refining that obtains purifying saves backup in 0-4 ℃;
3) fish sperm anti-freezing collection is preserved the preparation of liquid: first the refining after purifying is mixed with the volume ratio of 1:1 to 1:3 with the Hank's balance solution, whether the osmotic pressure that then uses osmometer to detect this mixed solution reaches target osmotic pressure, if osmotic pressure is higher or on the low side, reduces or add glucose and promote solution osmotic pressure to target osmotic pressure by directly add ultra-pure water to mixed solution.
2. method according to claim 1, is characterized in that, described target osmotic pressure is near the osmotic pressure of described fish refining or is beneficial to freeze proof osmotic pressure; Wherein, the osmotic pressure of fresh-water fishes spermatozoa preservative fluid is between 260-310mOs/kg, and the osmotic pressure of fish spermatozoa preservative fluid is between 150-200mOs/kg.
3. the fish sperm anti-freezing collection of an arbitrary described method preparation is preserved liquid, it is characterized in that, take Human seminal plasma as raw material, mixes making after purifying with the Hank's balance solution with the volume ratio of 1:1 to 1:3.
4. the described fish sperm anti-freezing of claim 3 collection is preserved the application of liquid in preventing the fish sperm cohesion.
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| Kajian Aglutinasi Spermatozoa Melalui Karakterisasi Plasma yang Dikoleksi dari Epididimis dan Seminalis Domba;Muhammad Haviz et al;《Journal Veteriner Desember》;20081231;第9卷(第4期);176-181 * |
| Muhammad Haviz et al.Kajian Aglutinasi Spermatozoa Melalui Karakterisasi Plasma yang Dikoleksi dari Epididimis dan Seminalis Domba.《Journal Veteriner Desember》.2008,第9卷(第4期),176-181. |
| 丁之德.哺乳动物精浆蛋白与精子成熟关系的研究进展.《国外医学计划生育分册2002年》.2002,第21卷(第1期),第2-5页. |
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| 哺乳动物精浆蛋白与精子成熟关系的研究进展;丁之德;《国外医学计划生育分册2002年》;20020215;第21卷(第1期);第2-5页 * |
| 王小花等.利用低温显微镜对斑马鱼精子最佳冷冻速率的筛选.《第七届全国低温生物医学及器械学术大会论文集》.2010,第25-31页. * |
| 鱼类精子膜脂检测方法的建立及其膜脂在精子超低温冷冻;吕刚等;《第七届全国低温生物医学及器械学术大会论文集》;20100730;第16-24页 * |
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