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Method for improving sperm motility and increasing in-vitro-fertilization rate of frozen bovine semen by aid of genistein

A technology of genistein and frozen semen, which is applied in animal delivery, veterinary instruments, medical science, etc., can solve the problems of low early embryonic development rate and lower semen quality, so as to improve the survival rate of linearly moving sperm and promote the development of early embryos. Development, anti-oxidative damage reduction effect

Active Publication Date: 2015-05-27
武汉市畜牧兽医科学研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0012] The present invention aims to solve the problems of reduced semen quality and low early embryo development rate after in vitro fertilization due to the increase of sperm active oxygen after thawing frozen bovine semen, and provides the use of genistein to improve the motility of frozen sperm and timely remove in vitro fertilization Excessive free radicals in the environment, thereby improving the method of bovine early embryo development rate and in vitro fertilization rate

Method used

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  • Method for improving sperm motility and increasing in-vitro-fertilization rate of frozen bovine semen by aid of genistein
  • Method for improving sperm motility and increasing in-vitro-fertilization rate of frozen bovine semen by aid of genistein
  • Method for improving sperm motility and increasing in-vitro-fertilization rate of frozen bovine semen by aid of genistein

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: The method of using genistein to improve sperm motility and in vitro fertilization rate of frozen bovine semen comprises the following steps in sequence: (1) Take a bovine frozen semen, thaw it and collect it into a centrifuge tube Centrifuge for the first time and discard the supernatant, then add it to the previously prepared SP-TALP washing solution according to the volume ratio of 1:1 and mix evenly, then mix the SP-TALP washing solution containing bovine semen with the concentration of 200 μmol / L Concentrated genistein stock solution (containing 1‰DMSO) was mixed evenly at a volume ratio of 19:1, so that the final concentration of genistein in the SP-TALP washing solution containing bovine semen was 10 μmol / L, and the final concentration of DMSO was 1‰, and then placed in a 37°C, 5% CO2 incubator to incubate for 2-3 hours, wherein the centrifugal speed of the first centrifugation is 1500-2000r / min, and the centrifugation time is 5-8min;

[0036] (2) P...

Embodiment 2

[0040] Embodiment 2: The in vitro fertilization method for improving sperm motility and in vitro fertilization rate of frozen bovine semen includes the following steps in turn: (1) Take a piece of bovine frozen semen, put it into a centrifuge tube after thawing, and first Centrifuge for a second time and discard the supernatant, then add it to the SP-TALP washing solution prepared in advance according to the volume ratio of 1:1, mix well, and put it in 37 ° C, 5% CO 2 Incubate in an incubator with 100% saturated steam for 2-3 hours; the centrifugal speed of the first centrifugation is 1500-2000r / min, and the centrifugation time is 5-8min;

[0041] (2) Same as embodiment one;

[0042] (3) The preparation in step (2) is to mix the IVF-TALP fertilization fluid containing sperm, the PHE capacitation fluid, and the concentrated genistein stock solution with a concentration of 200 μmol / L according to the volume ratio of 15.5:13:1.5, so that The concentration of genistein in the mix...

Embodiment 3

[0044] Embodiment 3: The in vitro fertilization method for improving sperm motility and in vitro fertilization rate of frozen bovine semen includes the following steps in turn: (1) Take a bovine frozen semen, put it into a centrifuge tube after thawing, and first Centrifuge for a second time and discard the supernatant, then add it to the SP-TALP washing solution prepared in advance according to the volume ratio of 1:1, mix well, and put it in 37 ° C, 5% CO 2 Incubate in the incubator for 2-3 hours; the centrifugation speed of the first centrifugation is 1500-2000r / min, and the centrifugation time is 5-8min;

[0045] Step (2), step (3) are all the same as embodiment one;

[0046] (4) Take 40-45 μL of IVF-TALP fertilization fluid to prepare fertilization drops, and put them in 38.5°C, 5% CO 2 Pre-incubate with 100% saturated steam incubator for 2 hours, add 5-6 bovine oocytes that have been matured in vitro, and add genistein concentrated stock solution with a concentration of...

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Abstract

The invention provides a method for improving the sperm motility and increasing an in-vitro-fertilization rate of frozen bovine semen by the aid of genistein. The method is particularly characterized in that the genistein containing 1%o of DMSO (dimethyl sulfoxide) is added into SP-TALP (serum phosphate-total alkaline phosphatase) washing liquid for washing the frozen bovine semen in earlier-stage processing for the frozen bovine semen, or mixed liquid of PHE (phenylalanine) capacitation liquid and IVF-TALP (in vitro fertilization-total alkaline phosphatase) fertilization liquid containing bovine semen in a capacitation processing stage of second-stage pre-fertilization semen or prepared fertilization drops containing oocytes in a third-stage in-vitro-fertilization procedure; the added amounts of the genistein in two earlier stages of a fertilization procedure of each bottle of frozen bovine semen are 10 micromoles per liter, and the final concentration of the genistein in mixed liquid of the bovine semen and the fertilization drops containing the oocytes is 10 micromoles per liter when the genistein is added in a third stage of the fertilization procedure of each bottle of frozen bovine semen. The method has the advantages that the genistein is respectively used in the three in-vitro-fertilization stages, so that the sperm motility is greatly improved, and the sperm fertilization rate is greatly increased.

Description

technical field [0001] The invention relates to an active substance and a method for increasing the sperm motility and in vitro fertilization rate of bovine frozen semen, belonging to the technical field of livestock embryo engineering, in particular to a method for improving the motility and in vitro fertilization rate of bovine frozen semen sperm by using genistein . Background technique [0002] At present, the frozen semen of cattle has entered commercial production and is widely used in artificial insemination technology. The freezing and thawing process in semen production and use destroys the dynamic balance of reactive oxygen species (ROS) in sperm under normal physiological conditions, leading to an increase in sperm ROS, causing changes in the fluidity of sperm membranes, resulting in oxidative damage, which affects Sperm motility, plasma membrane integrity and morphology, etc., reduce the fertilization ability. At the same time, ROS can damage lipids, nucleic ac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61D19/02
Inventor 程蕾陈洪波胡修忠辛友东凌明湖刘晓华高扬泽王定发夏瑜
Owner 武汉市畜牧兽医科学研究所
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