CN101831383B - Method and device for separating sperms - Google Patents
Method and device for separating sperms Download PDFInfo
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- CN101831383B CN101831383B CN2010101795775A CN201010179577A CN101831383B CN 101831383 B CN101831383 B CN 101831383B CN 2010101795775 A CN2010101795775 A CN 2010101795775A CN 201010179577 A CN201010179577 A CN 201010179577A CN 101831383 B CN101831383 B CN 101831383B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- Centrifugal Separators (AREA)
Abstract
The invention discloses simple and high-efficient method and device for separating sperms. The device consists of an Eppendorff centrifugal tube, a separating tube and a rubber ring. The method comprises the steps of: reasonably controlling the level height of culture solution; and culturing in a 5 percent CO2 culture container at 37 DEG C for 15minutes for separating the sperms. The concentration of the sperms can be adjusted by adopting a low-speed centrifugal method; about 95 percent of obtained sperms have athletic ability; and the collected solution generally has no blood cells. The invention is suitable for an in vitro capacitation experiment.
Description
Technical field
The present invention relates to a kind of method and device of separated sperm.
Background technology
Fertilization is the process that sperm and ovum combine, and is a kind of important biological phenomena.Mammiferous sperm can not combine with ovum after just having penetrated, and must after jenny reproduction pipeline stays for some time, perhaps in suitable nutrient solution, behind the cultivation certain hour, just have and ovum bonded ability, and this process is called capacitation.Capacitation is one of natural reproduction very the key link.At present, capacitation mechanism is not illustrated as yet, and most of data are in the suitable nutrient solution of external use, to cultivate sperm to obtain.Mouse and rat are that more animal is used in the research capacitation, and the sperm of generally getting mouse or rat epididymis afterbody carries out the capacitation Mechanism Study.The problem that exists now has two.1. when cauda epididymis was got sperm, the little blood vessel on epididymis surface was difficult to reject fully, in the sperm solution that obtains the part hemocyte is always arranged.2. a small amount of death or the very poor sperm of motor capacity are arranged in the sperm solution.Hemocyte, dead sperm and the very poor sperm of motor capacity have certain influence to the capacitation Study on Mechanism.In order to remove the hemocyte in the sperm nutrient solution, dead sperm and the very poor sperm of motor capacity, generally adopt upper reaches method (swim up), be about to sperm and be placed on 37 ℃ of 5% CO in the centrifuge tube
2Cultivate certain hour, get the sperm in the nutrient solution of upper strata then.Behind conventional upper reaches method processing sperm, still there is the part hemocyte in the nutrient solution of upper strata sometimes, the percentage that the motor capacity sperm is arranged is generally at 80-90%.
The contrive equipment of disclosed separated sperm also has " a kind of double tube type pelagic screening sperm tube " (patent No. 200920019260.8), comprises two standpipes, and first standpipe is through the transverse tube and the second standpipe UNICOM; On the lower sides of first standpipe annular lug is set, the projection sidewall of annular lug and the angle of horizontal plane are α, 60 degree≤α≤85 degree; The equal diameters of first standpipe and second standpipe; All be D, the diameter of transverse tube is d, 2d=D; The making of this device is more complicated than apparatus of the present invention, and is impracticable.In order to obtain high-quality sperm; We have designed a kind of device of simple and practical separated sperm; And found out the good schedule of operation of separating effect, and the percentage that the motor capacity sperm is arranged in the sperm of acquisition is about 95%, and collecting in the liquid does not generally have hemocyte.
Summary of the invention
The object of the present invention is to provide a kind of method and device thereof of simple, sharp separation sperm, operation steps of the present invention is suitable in the 1.5ml centrifuge tube, operating all with a small amount of sample.This method is particularly suitable for the screening of mouse and rat sperm, for the capacitation Study on Mechanism provides high-quality sperm.
A kind of device of separated sperm; Described device is made up of 1.5ml Eppendorff centrifuge tube, separator tube and rubber ring; Described separator tube is in centrifuge tube inside, and is the doline that communicates up and down, and the internal diameter of its bottom opening is 2mm; Open-topped internal diameter is 6mm, and the length of separator tube is 14-16mm; Described rubber ring be wound on the outer wall of separator tube and with the distance of separator tube bottom be the 1/3-1/2 of separator tube total length, the inwall that rubber ring is close to centrifuge tube makes the outer wall of described separator tube and the inwall of bottom and centrifuge tube all leave the space.
Use the method for above-mentioned device separated sperm, may further comprise the steps:
(1) gets the Eppendorff centrifuge tube bottom that 0.2ml sperm solution is placed on 1.5ml;
(2) separator tube is placed in the centrifuge tube, rubber ring on the separator tube and centrifuge tube inwall are adjacent to;
(3) nutrient solution is added in the centrifuge tube through separator tube, the nutrient solution liquid level is higher than separator tube top 1mm;
(4) cover the centrifuge tube lid gently, centrifuge tube is placed 37 ℃ of 5%CO
2Cultivate 15min in the incubator;
The separator tube outer wall and the nutrient solution between the centrifuge tube that (5) will be in rubber ring top with pipettor are transferred in the new centrifuge tube, and the centrifugal 10min of 300g abandons supernatant, with the resuspended sperm of nutrient solution, regulate sperm to desired concn.
This separation device is made up of 1.5ml Eppendorff centrifuge tube, separator tube and rubber ring, and the inwall that rubber ring is close to centrifuge tube makes the outer wall of described separator tube and the inwall of bottom and centrifuge tube all leave the space.The sperm that is placed on the centrifuge tube bottom has only the separator tube of entering bottom opening, is upward through separator tube, could arrive the space that is surrounded by separator tube outer wall, centrifuge tube inwall and rubber ring.The nutrient solution liquid level only is higher than separator tube top 1mm, makes the sperm that gets in the space from the separator tube open top be difficult for returning in the separator tube.The separator tube bottom opening is less, and internal diameter is 2mm, does not have the sperm of motor capacity to be difficult for getting into the separator tube bottom opening, and separator tube is the doline that communicates up and down, and doline makes sperm move upward easily and is not easy to move downward.Apparatus of the present invention and method are simple to operate, and separated sperm is effective, and the percentage that the motor capacity sperm is arranged in the sperm of acquisition exceeds 5-10% than use one type of prior art syringe and method about 95%, and generally do not have hemocyte in the collection liquid.
Description of drawings
Fig. 1 is a device synoptic diagram of the present invention;
1 is centrifuge tube among the figure; 2 is separator tube, and 3 is rubber ring.
Embodiment
Further specify the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1
The device of separated sperm of the present invention; Form by 1.5ml Eppendorff centrifuge tube 1, separator tube 2 and rubber ring 3; Separator tube 2 is in centrifuge tube 1 inside; And be the doline that communicates up and down, the internal diameter of its bottom opening is 2mm, and open-topped internal diameter is 6mm; Rubber ring 3 be wound on the outer wall of separator tube 2 and with the distance of separator tube 2 bottoms be the 1/3-1/2 of separator tube 2 total lengths, the inwall that rubber ring 3 is close to centrifuge tube 1 makes the outer wall of described separator tube 2 and the inwall of bottom and centrifuge tube 1 all leave the space.
The process that the employing said apparatus carries out separated sperm is following:
(1) gets the Eppendorff centrifuge tube bottom that 0.2ml sperm solution is placed on 1.5ml by ordinary method.
(2) the sperm separator tube is placed in the centrifuge tube, rubber ring on the separator tube and centrifuge tube inwall will be adjacent to.
(3) the BWW nutrient solution is added in the centrifuge tube through separating the mouth of pipe, the nutrient solution liquid level is approximately higher than separator tube top 1mm.
(4) cover the centrifuge tube lid gently, centrifuge tube is placed 37 ℃ of 5%CO
2Cultivate 15min in the incubator.
(5) nutrient solution between separator tube outer wall and the centrifuge tube is transferred in the new centrifuge tube, the centrifugal 10min of 300g abandons supernatant, with the resuspended sperm of BWW nutrient solution, regulates sperm to suitable concn.The percentage that in the sperm that obtains the motor capacity sperm is arranged is about 95%, and collecting in the liquid does not generally have hemocyte.
Claims (2)
1. the device of a separated sperm; It is characterized in that described device is made up of 1.5mL Eppendorf centrifuge tube, separator tube and rubber ring, described separator tube is in centrifuge tube inside; And be the doline that communicates up and down; The internal diameter of its bottom opening is 2mm, and open-topped internal diameter is 6mm, and the length of separator tube is 14-16mm; Described rubber ring be wound on the outer wall of separator tube and with the distance of separator tube bottom be the 1/3-1/2 of separator tube total length, the inwall that rubber ring is close to centrifuge tube makes the outer wall of described separator tube and the inwall of bottom and centrifuge tube all leave the space.
2. use the method for the described device separated sperm of claim 1, may further comprise the steps:
(1) gets the Eppendorf centrifuge tube bottom that 0.2mL sperm solution is placed on 1.5mL;
(2) separator tube is placed in the centrifuge tube, rubber ring on the separator tube and centrifuge tube inwall are adjacent to;
(3) nutrient solution is added in the centrifuge tube through separator tube, the nutrient solution liquid level is higher than separator tube top 1mm;
(4) cover the centrifuge tube lid gently, centrifuge tube is placed 37 ℃ of 5%CO
2Cultivate 15min in the incubator;
The separator tube outer wall and the nutrient solution between the centrifuge tube that (5) will be in rubber ring top with pipettor are transferred in the new centrifuge tube, and the centrifugal 10min of 300g abandons supernatant, with the resuspended sperm of nutrient solution, regulate sperm to desired concn.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101795775A CN101831383B (en) | 2010-05-21 | 2010-05-21 | Method and device for separating sperms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101795775A CN101831383B (en) | 2010-05-21 | 2010-05-21 | Method and device for separating sperms |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101831383A CN101831383A (en) | 2010-09-15 |
| CN101831383B true CN101831383B (en) | 2012-05-02 |
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ID=42715603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101795775A Expired - Fee Related CN101831383B (en) | 2010-05-21 | 2010-05-21 | Method and device for separating sperms |
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| CN (1) | CN101831383B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436428B (en) * | 2013-07-19 | 2015-02-11 | 浙江大学 | Automatic sperm floating apparatus and use method thereof |
| CN110918149A (en) * | 2018-09-20 | 2020-03-27 | 牛尾电机(苏州)有限公司 | Centrifugal tube |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2539628B1 (en) * | 1983-01-25 | 1988-11-10 | Fondation Rech Hormonologie | PROCESS FOR SEPARATING MOBILE CELLS, ESPECIALLY MOBILE SPERMATOZOIDS, AND A DEVICE FOR CARRYING OUT SAID METHOD |
| US5686302A (en) * | 1993-02-09 | 1997-11-11 | Zech; Josef | Device for removing sperm cells form seminal fluid |
| CN1312855A (en) * | 1998-08-14 | 2001-09-12 | 基因诊断有限公司 | Separation and detection of spermatozoa |
| CN2790415Y (en) * | 2005-04-13 | 2006-06-28 | 阎沈军 | Filter type sperm separator |
| CN201695034U (en) * | 2010-05-21 | 2011-01-05 | 中南大学 | Device for separating sperms |
-
2010
- 2010-05-21 CN CN2010101795775A patent/CN101831383B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2539628B1 (en) * | 1983-01-25 | 1988-11-10 | Fondation Rech Hormonologie | PROCESS FOR SEPARATING MOBILE CELLS, ESPECIALLY MOBILE SPERMATOZOIDS, AND A DEVICE FOR CARRYING OUT SAID METHOD |
| US5686302A (en) * | 1993-02-09 | 1997-11-11 | Zech; Josef | Device for removing sperm cells form seminal fluid |
| CN1312855A (en) * | 1998-08-14 | 2001-09-12 | 基因诊断有限公司 | Separation and detection of spermatozoa |
| CN2790415Y (en) * | 2005-04-13 | 2006-06-28 | 阎沈军 | Filter type sperm separator |
| CN201695034U (en) * | 2010-05-21 | 2011-01-05 | 中南大学 | Device for separating sperms |
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| Publication number | Publication date |
|---|---|
| CN101831383A (en) | 2010-09-15 |
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