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Method for improving moveability of sperm in frozen bull semen and in vitro fertilization rate

A technology of freezing semen and in vitro fertilization, which is applied in animal delivery, medical science, veterinary equipment, etc., and can solve the problems of reduced semen quality and low early embryonic development rate

Active Publication Date: 2013-12-18
武汉市畜牧兽医科学研究所
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  • Abstract
  • Description
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Problems solved by technology

[0015] 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 a method that can improve the motility of frozen sperm by adding biologically active substances 1. Timely removal of excessive free radicals in the environment of in vitro fertilization, thereby improving the method for the early embryonic development rate of cattle

Method used

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  • Method for improving moveability of sperm in frozen bull semen and in vitro fertilization rate
  • Method for improving moveability of sperm in frozen bull semen and in vitro fertilization rate
  • Method for improving moveability of sperm in frozen bull semen and in vitro fertilization rate

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Embodiment 1

[0048] Embodiment 1: The method for improving sperm motility and in vitro fertilization rate of bovine frozen semen comprises the following steps in turn: (1) Take a piece of bovine frozen semen, collect it in a centrifuge tube after thawing and centrifuge for the first time And discard the supernatant, then add in the previously prepared SP-TALP washing solution according to the volume ratio of 1:1 and mix evenly, then the SP-TALP washing solution containing bovine semen, the glutathione concentration of 100mmol / L The concentrated stock solution and the caffeine concentrated stock solution with a concentration of 100mmol / L were mixed evenly according to the volume ratio of 18:1:1, so that the final concentrations of glutathione and caffeine in the SP-TALP washing liquid containing bovine semen were both 5mmol / L L, then put into 37℃, 5%CO 2 and 100% saturated steam incubator for 2-3 hours; wherein the centrifugal speed of the first centrifugation is 1500-2000r / min, and the cen...

Embodiment 2

[0053] Embodiment 2: The method for improving sperm motility and in vitro fertilization rate of bovine frozen semen comprises the following steps in turn: (1) Take a piece of bovine frozen semen, put it into a centrifuge tube for the first centrifugation after thawing 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;

[0054] (2) Same as embodiment one;

[0055] (3) The IVF-TALP fertilization fluid containing sperm, PHE capacitation fluid, glutathione concentrated stock solution with a concentration of 100mmol / L and caffeine concentrated stock solution with a concentration of 100mmol / L were prepared in step (2). The solution was mixed evenly according to the volume ratio of 14:13:1.5:1.5, so t...

Embodiment 3

[0057] Embodiment 3: The method for improving sperm motility and in vitro fertilization rate of bovine frozen semen comprises the following steps in turn: (1) Take a piece of bovine frozen semen, put it into a centrifuge tube after thawing and centrifuge for the first time 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;

[0058] Step (2), step (3) are all the same as embodiment one;

[0059] (4) Take 40-45 μL of IVF-TALP fertilization fluid to prepare fertilization drops, and put them in 38.5°C, 5% CO 2 and 100% saturated steam incubator for 2 hours, add 5-6 bovine oocytes that have been matured in vitro to make the total volume 48μL, and add 100mmol / L oocytes at a volume ratio of 8:1:1 Glu...

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Abstract

The invention provides a method for improving the moveability of sperm in frozen bull semen and the in vitro fertilization rate. The method is characterized in that when SP-TALP washing liquid is used for washing at a prior disposal stage of the frozen bull semen, or when PHE (phenylalanine) capacitation liquid is used for capacitation disposal at a capacitation disposal stage of the semen before fertilization, or when fertilization drips are prepared at an in vitro fertilization stage, glutathione and caffeine of which the purities are both higher than 98% are added; in the fertilization process of each piece of frozen bull semen, in the SP-TALP washing liquid containing bull semen or in the mixed liquid of IVF-TALP seminal fluid containing the bull semen and the PHE capacitation liquid or in the mixed liquid of the fertilization drips containing oocytes and the bull semen, the final concentration of the glutathione and the caffeine is 5mmol / L. According to the method disclosed by the invention, two substances are jointed to be respectively applied to three stages in the fertilization process of the frozen bull semen, the moveability of the sperm is greatly improved, and the fertilization rate of the sperm is improved.

Description

technical field [0001] The invention belongs to the technical field of livestock embryo engineering, and in particular relates to a method capable of improving sperm motility and in vitro fertilization rate of bovine frozen semen by adding biologically active substances. Background technique [0002] At present, the frozen semen of cattle has entered commercial production and is widely used in artificial insemination technology. However, 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 peroxidation of sperm membranes and DNA damage, thereby It affects sperm motility, plasma membrane integrity and fertilization potential, and reduces the quality of semen. [0003] Glutathione (GSH) is an important non-protein sulfhydryl compound in mammalian cells. As one of the substrates of the peroxidase-reductase system, it...

Claims

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Application Information

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IPC IPC(8): A61D19/02
Inventor 程蕾陈洪波刘晓华高扬泽辛友东王定发胡修忠凌明湖夏瑜
Owner 武汉市畜牧兽医科学研究所
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