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Application of 1,1,2,2-tetra-[4-(2-triethylethoxyamino)]styrene tetrabromide in sperm separation

A technology based on triethyl ethoxyamine and styrene, applied in the field of sperm separation, can solve the problems of aggregation fluorescence quenching, unsatisfactory sperm separation effect, and sudden drop in fluorescence intensity.

Active Publication Date: 2015-09-16
大连金弘基种畜有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In related technologies, the fluorescent dyes commonly used in the above methods are prone to aggregation fluorescence quenching: when the concentration of the dye is high, the dye molecules will aggregate and cause a sudden drop in the emitted fluorescence intensity
When the density of semen to be separated is high, a higher concentration of fluorescent dye is required for labeling, but the high concentration of dye molecules will aggregate, and then the aggregation fluorescence quenching will occur, resulting in unsatisfactory sperm separation effect

Method used

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  • Application of 1,1,2,2-tetra-[4-(2-triethylethoxyamino)]styrene tetrabromide in sperm separation

Examples

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Embodiment 1

[0011] This example provides the application of 1,1,2,2-tetrabromide 1,1,2,2-tetra-[4-(2-triethylethoxyamino)]styrene to sperm separation, including the following steps:

[0012] The first step: adding fluorescent dyes and staining talp to the semen to be separated, and then incubating to obtain a mixed solution.

[0013] Step 2: Use flow cytometry to separate the mixed solution to obtain separated X sperm and Y sperm.

[0014] Among them, the fluorescent dye is 1,1,2,2-tetrabromide 1,1,2,2-tetra-[4-(2-triethylethoxyamino)]styrene, and each semen to be separated contains 400 million sperm. , Add at least 7μg fluorescent dye.

[0015] The fluorescent dye used in the above application is 1,1,2,2-tetrabromide 1,1,2,2-tetra-[4-(2-triethylethoxyamino)]styrene (ie TTAPE), which is an aggregation-inducing dye Molecules that emit light, that is, when the concentration of the fluorescent dye is large or aggregated, the inner rotation of the fluorescent dye molecule is frozen or aggregates are...

Embodiment 2

[0047] The application of TTAPE in sperm separation includes the following steps:

[0048] First, set the temperature of the circulating water bath to 34℃ and preheat it 30 minutes in advance; place the 4% egg yolk talp solution in the water bath for 15 minutes before use; use a pipette to accurately suck the combined antibiotics (css) into the fresh Shake the semen in a centrifuge tube (the ratio is 1mL fresh original sperm to 20 μl css); accurately calculate the sperm density, the amount of semen used once, and the amount of staining talp solution; gradient staining is performed with a gradient of ≥34 μl.

[0049] Secondly, the total number of sperm in each sample tube is 400 million and the volume is 4 mL; use a micropipette to absorb fluorescent dye and add it to the sample tube, mark the serial number and batch; then add a certain amount of preheated staining talp into the sample tube Mix well; use a pipette to accurately pipette a certain amount of fresh semen into the sample...

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Abstract

The invention relates to the technical field of sperm separation, and specifically relates to an application of tetra-brominated 1, 1, 2, 2-tetra-[4-(2-triethyl ethoxy amido)] styrene to sperm separation. The application comprises the following steps: adding a fluorescent dye and a dyeing working fluid into a to-be-separated seminal fluid, and then, incubating to obtain a mixed liquid, wherein the fluorescent dye is tetra-brominated 1, 1, 2, 2-tetra-[4-(2-triethyl ethoxy amido)] styrene, and at least 7 micrograms of fluorescent dye is added when the to-be-separated seminal fluid contains 0.4 billion of sperms; separating the mixed liquid by a flow cytometry to obtain separated X sperms and Y sperms. According to the invention, tetra-brominated 1, 1, 2, 2-tetra-[4-(2-triethyl ethoxy amido)] styrene is applied to sperm separation. When the to-be-separated seminal fluid is higher in density and the high-concentration fluorescent dye is required, a fluorescence quenching phenomenon is avoided, so that the adverse effect on the separation effect is avoided.

Description

Technical field [0001] The present invention relates to the technical field of sperm separation, in particular, to the application of 1,1,2,2-tetrabromide 1,1,2,2-tetra-[4-(2-triethylethoxyamino)]styrene to sperm separation . Background technique [0002] The separation of animal X sperm and Y sperm by flow cytometry is a new way of animal sex control. Its working principle is to use the difference in DNA content in animal semen containing X chromosomes and Y chromosomes, dye with fluorescent dyes, and then The intensity of the fluorescence emitted is determined by the computer to determine the sperm category, and then by adding a charge to the semen droplets, according to the principle of static electricity, the semen containing the X chromosome and the semen of the Y chromosome are separated. [0003] In the related art, the fluorescent dyes commonly used in the above methods are prone to aggregate fluorescence quenching: when the concentration of the dye is high, the dye molecu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/076G01N15/14
Inventor 于建香帅志强苏冠宇武光明姚天宇
Owner 大连金弘基种畜有限公司
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