Fluorescent staining method for separating dairy cow sperms

A dyeing method, fluorescent dyeing technology, applied in the preparation of test samples, etc., to achieve the effects of improving quality, easy sorting and separation, and large difference in fluorescence intensity

Active Publication Date: 2010-08-04
内蒙古赛科星繁育生物技术(集团)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now, including the United States, the birthplace of this technology, the utilization rate of bulls using this technology is only about 30%.

Method used

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  • Fluorescent staining method for separating dairy cow sperms
  • Fluorescent staining method for separating dairy cow sperms
  • Fluorescent staining method for separating dairy cow sperms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] material:

[0046] 140 million milk cow essence (cow number: 11105825, original essence concentration 730 million / ml)

[0047] 2. Buffer composition:

[0048] HEPES, molecular formula C 6 H 12 N 2 O 4 S, concentration 9.52×10 -3 g / ml, molar concentration 40.00mM;

[0049] Magnesium chloride hexahydrate crystal, molecular formula MgCl 2 ·6H 2 O, concentration 0.08×10 -3 g / ml, the molar concentration is 0.40mM;

[0050] Sodium chloride, molecular formula NaCl, concentration 5.518×10 -3 g / ml, molar concentration 95.00mM;

[0051] Potassium chloride, molecular formula KCl, concentration 0.224×10 -3 g / ml, molar concentration 3.00mM;

[0052] Disodium hydrogen phosphate / anhydrous, molecular formula Na 2 HPO 4 , Concentration 0.04×10 -3 g / ml, molar concentration 0.30mM;

[0053] Sodium bicarbonate, molecular formula NaHCO 3 , Concentration 0.84×10 -3 / gml, molar concentration of 10.0mM;

[0054] Pyruvate, molecular formula NaPyruvate, concentration 0.22×10 -3 g / ml, molar concentration 2.00m...

Embodiment 2

[0071] material:

[0072] 140 million milk cow essence (cow number: 11103411, original essence concentration 920 million / ml)

[0073] 2. Separator model SX-Moflo, produced in the United States

[0074] Proceed as follows:

[0075] (1) Add the cow essence to a 15ml centrifuge tube, centrifuge for 5 minutes, aspirate and discard 0.5ml, 1ml, 1.5ml and 2ml of supernatant respectively, and complete the step of centrifugation to remove the seminal plasma from the cow semen;

[0076] (2) Fluorescence staining of cow sperm: mix 100 million sperm with part of seminal plasma removed: 5 microliters of fluorescent dye hochest33342, and bath at 34°C for 45 minutes;

[0077] (3) Separate stained bovine sperm by flow cytometry. The separation process is as follows:

[0078] a) Select the separation mode Purify 1;

[0079] b) Put the sample in the sample adding station, turn on the pressure and sample switch on the sampler, press boost to make the sample flow out of the tip, and adjust the sample pressure...

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PUM

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Abstract

The invention relates to a fluorescent staining method for separating dairy cow sperms, comprising the following steps of: centrifuging dairy cow semen before staining to remove part of seminal plasma, and mixing the semen after part of the seminal plasma is removed with fluorescent dye according to a certain proportion for staining. The novel fluorescent staining method has the advantages of good staining effect, great fluorescence intensity difference with X/Y sperms after staining, easy separation of sperms and the like, thereby not only improving the separating effect and efficiency of sperms, but also improving the use ratio of breeding oxen.

Description

Technical field [0001] The invention belongs to the field of cell and reproductive biotechnology. It is suitable for the use of flow cytometry for milk cow sperm separation and production control of frozen sperm. Background technique [0002] At present, sperm separation based on flow cytometry is an effective technology and means for gender control of dairy cows or beef cattle, and fluorescent staining of sperm is a necessary and key step in the separation process. Mammalian sex control is a subject that people have been interested in and conducted a lot of extensive research. After long-term research, it has been proved that sperm separation based on flow cytometry is the most successful and practical sex control technology. This technology has been successful in a variety of mammals, has been promoted and used in many countries including China, and has played or is playing a huge role in the sex control of dairy cows. Staining sperm with fluorescent dyes is a key step in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30
Inventor 周文忠李喜和王建国钱松晋
Owner 内蒙古赛科星繁育生物技术(集团)股份有限公司
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