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Method for improving efficiency of bovine embryo preparation in vitro

A technology of preparation efficiency and embryo, applied in the field of cell biology and molecular biology, can solve the problem of lack of research on the embryo development rate of MM102, and achieve the effect of high blastocyst rate

Pending Publication Date: 2018-09-28
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, MM102 has not been studied in terms of improving the rate of embryonic development

Method used

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  • Method for improving efficiency of bovine embryo preparation in vitro
  • Method for improving efficiency of bovine embryo preparation in vitro
  • Method for improving efficiency of bovine embryo preparation in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The in vitro collection and in vitro maturation culture of embodiment 1 bovine oocyte

[0050]Collect the ovaries of slaughtered cattle from the slaughterhouse, store them in a clean insulation bucket filled with 0.9% sterilized saline, and transport them back to the laboratory within 2-4 hours. After the ovaries are delivered to the laboratory, they are washed at least 3 times in 0.9% sterile saline at 20-25°C. The bovine ovary oocytes are collected using the syringe extraction method, and the follicles with a diameter of 2-7mm on the surface of the ovary are collected with a syringe. to extract. Collect the cumulus-oocyte complexes (COCs) with uniform cytoplasm and multiple layers of complete granulosa cells from the extracted follicular fluid under a stereomicroscope. in vitro maturation culture. All COCs were matured in vitro in four-well plates. The culture system is 700 μL / well of in vitro maturation culture medium, and 300 μL / well of paraffin oil is spread on ...

Embodiment 2

[0053] The in vitro fertilization of embodiment 2 bovine oocyte

[0054] After the oocytes are matured in vitro and cultured for 22-24 hours, the oocytes are fertilized in vitro. For the purchased bull semen, thaw the sperm in a 15mL sterile centrifuge tube containing 8mL of in vitro fertilization solution A according to the ratio of the number of eggs to the number of semen tubes of 100:1. Centrifuge at 37°C, 4000 rpm for 5 minutes. Wash twice, after sucking off the supernatant, slowly add 1-2mL in vitro fertilization culture medium to the surface of the sperm sedimentation, float in a water bath at 37°C for 3-5min, absorb the turbid part of the upper layer into a 1.5mL centrifuge tube, add etc. Volume of in vitro fertilization solution B, after mixing evenly, add fertilization drops, the concentration of sperm in each drop of fertilization drops is 1×10 6 -5×10 6 individual / mL. After in vitro maturation cultured COCs were washed 3 times with in vitro fertilization soluti...

Embodiment 3

[0059] The in vitro development and cultivation of embodiment 3 bovine fertilized eggs

[0060] After in vitro fertilization and culture of sperm and oocytes for 6 hours, the fertilized eggs were transferred into the in vitro development medium, and gently tapped repeatedly with a 100 μL pipette to remove the granulosa cells and the sperm adhering to the fertilized eggs. Liquid wash 3 times. Transfer the fertilized eggs into IVC-1 droplets for in vitro development, 20 pieces / drop, and put the petri dish into the incubator for development and culture for 48 hours. The culture system is 50 μL per drop of in vitro development culture medium, the surface of the liquid layer is covered with paraffin oil, and CO is added before in vitro development. 2 Balance in the incubator for more than 5 hours; culture in vitro until 48 hours, count the number of cleavages, wash the cleaved embryos with IVC-2 solution containing different concentrations of MM102 for 3 times, put them into the c...

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Abstract

The invention provides a method for improving the efficiency of bovine embryo preparation in vitro. The method comprises the steps that in vitro collection of bovine oocyte is performed, in vitro maturation culture of oocyte-cumulus complex is performed, in vitro fertilization of bovine oocyte is performed and ectogenesis culture is performed. The culture solution used in the ectogenesis culture contains 0.01-70[um]M of C35H49F2N7O4.CF3CO2H (MM102). Under external conditions, a best bovine in vitro fertilization system is selected, after 48 hours of in vitro fertilization, blastomere is placedin the development fluid with different concentrations of MM102, the results show that the MM102 can promote the ectogenesis of bovine fertilized eggs, and higher blastocyst rate can be obtained. Themethod can be used for improving the efficiency of bovine in vitro embryo preparation. The method for improving the efficiency of bovine in vitro embryo preparation provides certain theoretical basisfor the research of in vitro fertilization technology of livestock and can be further applied in the fields such as livestock reproduction and genetic seed preservation of livestock.

Description

technical field [0001] The invention relates to the fields of cell biology and molecular biology, in particular to a method for improving the efficiency of bovine embryo preparation in vitro. Background technique [0002] Bovine in vitro fertilization technology includes in vitro collection and maturation of oocytes, in vitro sperm capacitation, in vitro fertilization of sperm eggs, and in vitro culture of fertilized eggs. In 1977, Iritane and Niwa obtained the world's first in vitro fertilized cattle. Research on in vitro fertilization in our country started in the late 1980s. On the basis of previous studies, our country has made rapid progress. In 1989, academician Xurigan and others successfully bred my country's first in vitro fertilized cattle. Since then, the research teams of Fan Biqin, Lu Kehuan, Shi Deshun and others have successively obtained in vitro fertilized cattle. So far, the offspring of most domesticated animals have been successfully obtained due to th...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N5/075A61D19/00
CPCA61D19/00C12N5/0604C12N5/0609C12N2500/05C12N2500/30C12N2500/32C12N2500/34C12N2500/35C12N2500/80C12N2501/31
Inventor 李雪玲韩雪洁王晨周正伟
Owner INNER MONGOLIA UNIVERSITY
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