Two-layer percoll density gradient centrifugal separation method of boar sperms

A density gradient centrifugation, density gradient technology, applied in animal cells, vertebrate cells, germ cells, etc., can solve the problems of affecting sperm fertilization ability, difficult to remove sperm, low sperm motility, etc., to improve poor semen liquefaction and improve pregnancy. rate effect

Inactive Publication Date: 2011-11-23
JILIN UNIV
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Problems solved by technology

The current problems are: the upstream sperm are easy to cluster and affect the fertilization ability of sperm [2] ; low motility of retrieved sperm [3] ; Presence of antisperm antibodies in seminal plasma affects sperm motility and sperm cap

Method used

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  • Two-layer percoll density gradient centrifugal separation method of boar sperms
  • Two-layer percoll density gradient centrifugal separation method of boar sperms
  • Two-layer percoll density gradient centrifugal separation method of boar sperms

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Embodiment 1

[0017] Embodiment 1 The pig semen two-layer percoll density gradient centrifugation method provided by the present invention, the steps and conditions are as follows:

[0018] I. Preparation of Percoll: Commercially available Percoll has a volume concentration of 100%. It needs to be mixed with 1.5M sperm washing solution to reach physiological osmotic pressure, and then diluted to the required concentration with sperm washing solution.

[0019] (1) Preparation of percoll solution with a specific gravity of 1.113g / mL and a volume concentration of 90%: Take 45mL of commercially available Percoll, add 5mL of sperm washing solution, mix well, discard 3.75mL, add 3.75mL of water, mix After uniformity, filter with a 0.22 micron filter membrane for later use;

[0020] (2) The preparation of the percoll solution with a specific gravity of 1.066g / mL and a volume concentration of 45%: use an equal volume of percoll with a volume concentration of 90% to mix with the sperm washing liquid...

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Abstract

The invention provides a two-layer percoll density gradient centrifugal separation method of boar sperms. The method is suitable for normal sperm specimen and sperm specimen with low sperm density or low sperm motility, and is a simple, convenient and fast sperm treatment method. The method can be used for remove leucocytes, bacteria, fragments and anti-sperm antibodies in the sperms and improving the delayed sperm liquefaction, the sperm in-vitro capacitation and the like, so that the sperms can effectively fertilize ova to improve the pregnancy rate, and the percentage of the sperms with motility and normal shape in the obtained sperms is about 95%. The method provided by the invention is suitable for screening sperms with motility from fresh boar sperms and diluted sperms, thus providing high-quality sperms for the study of boar sperm capacitation mechanism and in-vitro fertilization.

Description

technical field [0001] The invention relates to a method for separating high-motility sperm from pig semen, in particular to a second-layer percoll density gradient centrifugal separation method of pig sperm. Background technique [0002] Fertilization is the process of fusion of egg and sperm into a zygote, which is the basic feature of sexual reproduction. With the development of embryo engineering and assisted reproductive technology, in vitro fertilization technology has been applied more and more. To complete in vitro fertilization, it is first necessary to obtain high-motility sperm in vitro. The sperm separation method mainly separates motile sperm from inactive sperm. Currently, the methods used include sperm upstream method, albumin column separation method, glass fiber filtration technology, and Percoll density gradient separation method, etc. Due to the immature technology of pig frozen semen, sperm are mainly obtained from fresh pig sperm dilutions [1] . Pig ...

Claims

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Application Information

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IPC IPC(8): C12N5/076
Inventor 李子义唐博高飞刘丽梅花明殷玉鹏马馨关继羽宋光启王中伟张鹏孔德龙
Owner JILIN UNIV
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