Novel bovine oocyte in vitro maturation culture solution

A technology for in vitro mature culture and oocyte, applied in tissue culture, microorganisms, biochemical equipment and methods, etc., can solve the problems of low in vitro fertilization rate and blastocyst formation rate, and high oocyte quality requirements

Active Publication Date: 2011-11-09
北京科润维德生物技术有限责任公司
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AI Technical Summary

Problems solved by technology

[0002] Since the 1930s, many scientific researchers have done a lot of work in the field of bovine oocyte growth and development in vitro. Mother cell culture conditions have been studied extensively and deeply, and great progress has been made. At present, the bovine oocyte maturation in vitro (invitro matrue, IVM) culture technology system has been established, but due to the oocyte maturation mechanism, maturation regulation There are still many problems that have not yet been clarified and some problems need to be solved urgently, so that the current method has high requirements for oocyte quality, low in vitro fertilization rate and blastocyst formation rate, etc.

Method used

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  • Novel bovine oocyte in vitro maturation culture solution
  • Novel bovine oocyte in vitro maturation culture solution

Examples

Experimental program
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Effect test

Embodiment 1

[0016] 1. In vitro maturation and culture method of oocytes (supernatant of amniotic epithelial cell culture medium is used as IVM medium supplement)

[0017] 1.1 Amniotic membrane epithelial cell culture supernatant-in vitro maturation (AECS-IVM) supplement and medium preparation

[0018] 1.1.1 Approximately 1mm 3 Cut the cattle or human amniotic membrane into pieces, and shake them in D-hank’s containing 0.125% Trypsins at 37°C for 30 minutes at a speed of 400-600 rpm;

[0019] 1.1.2 Filter the suspension with a cell sieve to collect amniotic epithelial cells;

[0020] 12.1.3 According to the conventional concentration (the number of cells is 10 5 ~10 6 A / bottle) inoculate amniotic membrane epithelial cells at 75cm 2 Culture flask

[0021] 1.1.4 Culture amniotic epithelial cells in vitro, and take the culture supernatant within 5-7 days;

[0022] 1.1.5 Centrifuge to remove cells and cell debris, 4℃, 1500-2500rpm, 15-30 minutes;

[0023] 1.1.6 Take the supernatant liquid, remove the sedi...

Embodiment 2

[0046] 1. In vitro maturation and culture method of oocytes (amniotic epithelial cells are used as IVM medium supplement)

[0047] 1.1 Culturing amniotic epithelial cells as IVM trophoblasts

[0048] 1.1.1 Approximately 1mm 3 Cut the amniotic membrane into pieces and shake it for 30 minutes at 37°C with 0.125% Trypsins D-hank’s at a speed of 400~600rpm

[0049] 1.1.2 Filter the suspension with a cell sieve to collect amniotic epithelial cells

[0050] 1.1.3 Inoculate amniotic epithelial cells at 75 cm at a regular concentration 2 Culture flask

[0051] 1.1.4 Culture bovine or human amniotic epithelial cells in vitro, pour out the supernatant, and add 100ml IVM medium (M199+10%FBS+10μg / ml FSH+0.1μg / ml LH+1μg / ml E 2 ), as a culture system of amniotic epithelial cells-in vitro maturation (AEC-IVM)

[0052] 1.2 In vitro culture of immature bovine eggs

[0053] 1.2.1 Routine collection and collection of bovine oocytes;

[0054] 1.2.2 After fully equilibrating the bovine oocytes in AEC-IVM mediu...

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Abstract

The invention relates to a novel bovine oocyte in vitro maturation (IVM) culture solution, belonging to the field of embryo biotechnolory. In the invention, the routine IVM culture solution is added with amniotic epithelia or the supernatant of the culture solution thereof to establish a novel bovine immature oocyte IVM culture system, so as to improve the quality and maturation rate of the IVM bovine oocyte, thus solving the problem of low fertilization rate and low formation rate of blastula in the existing culture method.

Description

Technical field [0001] The invention relates to the field of embryo engineering, in particular to an in vitro maturation fluid for bovine oocytes. Background technique [0002] As one of the foundations of embryonic bioengineering research, bovine oocyte in vitro maturation and culture technology. Since the 1930s, many scientific researchers have done a lot of work in the field of bovine oocyte growth and development in vitro. Extensive and in-depth studies have been carried out on the culture conditions of mother cells, and great progress has been made. At present, the invitro matrue (IVM) culture technology system of bovine oocytes has been established. However, due to oocyte maturation mechanism and maturity regulation There are still many unexplained and some urgent problems to be solved, so that the current methods have high requirements for oocyte quality, low in vitro fertilization rate and blastocyst formation rate. [0003] Amniotic membrane is formed on the 8th day of fe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06
Inventor 李荣旗
Owner 北京科润维德生物技术有限责任公司
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