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Sperm treating method

A treatment method, sperm technology, applied to other methods of inserting foreign genetic materials, using micro-injection, recombinant DNA technology, etc., can solve the problem of unsatisfactory head-to-tail separation

Inactive Publication Date: 2011-04-06
SHANGHAI INST OF PLANNED PARENTHOOD RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has also been reported that mouse sperm are treated with low-concentration alkaline solution to separate the head and tail of the sperm, but the effect of head and tail separation is not ideal, and the tail-docking rate is generally only about 10%.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Effect of NaOH on Separation of Head and Tail of Sperm in Mice

[0084] The inventor treated mouse epididymis tail sperm with 5-50mM NaOH solution at 0-37°C, observed under a microscope, and found that after 30 minutes of treatment with 5mM NaOH concentration, the sperm's activity rate had dropped to 0, and only a few Sperm are still swimming. See figure 1 .

[0085] The results show that with the increase of NaOH concentration, the efficiency of head-to-tail separation is gradually increasing, but different treatment temperatures have a significant impact on the effect of sperm head-to-tail separation. At 0-4°C, the effect of sperm head-to-tail separation is significantly lower than that under other temperature conditions .

Embodiment 2

[0087] Effect of NaOH on the Integrity of Mouse Sperm Plasma Membrane

[0088] The inventor used Transgreen / PI to counterstain the sperm after NaOH treatment, observed under the fluorescence microscope and detected its plasma membrane integrity with flow cytometry respectively ( figure 2 ), the experimental data show that ( image 3 ).

[0089] The results showed that 90% of the sperms with plasma membrane damage were treated with 5mM NaOH for 30 minutes, and the plasma membrane damage of sperm with a concentration of NaOH above 10mM was close to 100%.

Embodiment 3

[0091] Effects of NaOH on DNA Integrity of Mouse Sperm

[0092] With the increase of NaOH concentration, the degree of sperm DNA damage is greater. see results Figure 4 and Table 1.

[0093] Table 1 Effect of NaOH concentration and incubation temperature on mouse sperm DNA Impact

[0094]

[0095] * P<0.05, compared with the negative control group

[0096] The results showed that at 37°C, even 10mM NaOH had significant damage to sperm DNA, and low temperature could reduce the effect of NaOH on sperm DNA. At 0°C, 50mM NaOH incubated sperm for 30 minutes, and no significant damage occurred to sperm DNA .

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Abstract

The invention provides a sperm treating method. The method comprises the following step of: (1) hatching sperm suspension at the temperature of between 0 and 4DEG C for 0.5 to 3 hours to obtain intracytoplasmic sperm injection (ICSI) sperms, wherein the sperm suspension contains sperms and sodium hydroxide (NaOH); and according to the total volume of the sperm suspension, the concentration of the sodium hydroxide is 5 to 30nM.

Description

technical field [0001] The invention relates to artificial assisted reproductive technology, in particular to a method for processing sperm. Background technique [0002] Intracytoplasmic sperm injection (ICSI) technology is an artificial assisted reproductive technology developed in the late 1980s. It is the process of injecting a sperm or germ cell directly into the cytoplasm of the egg with the help of a micromanipulator, so that the sperm and egg are combined to complete the fertilization process. The efficiency of using ICSI technology to prepare transgenic animals is higher than that of male pronucleus microinjection. Therefore, using sperm as a carrier, combined with ICSI technology, has opened up a simple and feasible technical route for the research of transgenic animals. [0003] Mouse sperm tails are long, and it is necessary to use physical or chemical methods to remove the sperm tails before ICSI, so that ICSI can be performed smoothly. Therefore, it is very i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89
Inventor 施惠娟李翀宋丽伟林菊芳贾晓峰史庭燕袁瑶王会徐彦牛志宏
Owner SHANGHAI INST OF PLANNED PARENTHOOD RES
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