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Simple in-vitro maturation culture method for oocytes

A technology for in vitro maturation and cultivation of oocytes, which is applied in the field of simple in vitro maturation and cultivation of oocytes, can solve the problems of expensive, inconvenient to carry instruments and equipment, unfavorable research and application work of grassroots units, and achieve lower research threshold and reduce dependent effect

Inactive Publication Date: 2011-08-17
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the in vitro maturation and culture of oocytes is still a laboratory technology, and the equipment required is still relatively expensive for ordinary production units or some grassroots scientific research units.
This is not conducive to grass-roots units to carry out related research and application work; and when scientific research units carry out technology promotion or services at the grass-roots level, it is not convenient to carry equipment

Method used

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  • Simple in-vitro maturation culture method for oocytes
  • Simple in-vitro maturation culture method for oocytes
  • Simple in-vitro maturation culture method for oocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Development of parthenogenetic embryos under different maturation conditions of bovine oocytes

[0026] 1.1 Main instruments and equipment

[0027] CO 2 Incubator, biochemical incubator, stereo microscope, fluorescence microscope, sealing machine, constant temperature plate, special light-proof sealed air bag for culturing embryos, air duct with 0.22μm microporous membrane filter, disposable plastic dish and four The orifice plates were all products of NUNC Company, the filters were purchased from Milipore Company, and the bovine ovaries were obtained from the slaughterhouse of Xi’an City.

[0028] 1.2 Preparation of reagents and culture medium

[0029] TCM-199, insulin-transferrin-selenium (Insulin-Transferrin-Selenium, ITS) (Gibco), bovine serum albumin (bovine serum albumin, BSA) (Amresco), fetal bovine serum (FBS) (HyClone ), newborn bovine serum (NBS) (Shandong Yinxiang Weiye Group Co., Ltd.), human gonadotropin (hMG) (Livzon Pharmaceutical Factory of Livzon ...

Embodiment 2

[0050] 2. Comparison of the maturation rate of goat oocytes under different culture conditions

[0051] 2.1 Main instruments and equipment

[0052] CO 2 The incubator, water bath, stereo microscope, sealing machine, light-proof sealed air bag for culturing embryos, air duct with 0.22μm microporous membrane filter, disposable plastic dish and four-hole plate are all products of NUNC. , The filter was purchased from Milipore Company, and the goat ovary was obtained from the slaughterhouse of Yulin City.

[0053] 2.2 Preparation of reagents and culture medium

[0054] The oocyte washing solution is PBS+10% (V / V) NBS+10mmol / LHEPES+10mg / L heparin sodium, and the others are the same as Example 1.2 of the embodiment.

[0055] 2.3 Collection of goat oocytes

[0056] The ovaries were collected from Yulin Cashmere Goat Slaughterhouse, immediately put into a thermos bottle filled with sterilized and preheated saline to 37°C, and sent back to the laboratory immediately (2-3 hours). T...

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Abstract

The invention discloses a simple in-vitro maturation culture method for oocytes, which comprises the following steps: A1, collecting oocytes; A2, preparing a closed air bag and performing in-vitro maturation culture of oocytes; and A3, performing parthenogenetic activation and in-vivo culture of oocytes, namely after 22 to 24 hours of in-vitro maturation culture, selecting mature oocytes discharging first polar bodies, activating by a chemical process in the 28th hour, culturing for 3 days in merogenesis culture solution, culturing for 4 days in blastaea culture solution, and observing and counting a blastaea rate after 7 to 9 days. When a self-prepared closed air bag system is used, the in-vitro culture of the oocytes and embryos can be accomplished by using a common biochemical culture tank or water bath pot, so the dependency on expensive instruments is reduced and a research doorsill is reduced. When the simple closed air bag is used for in-vitro culture of the oocytes, blastaea can be grown, and the in-vitro maturation rate, merogenesis rate and blastaea rate are higher than those of oocytes cultured in a normal CO2 culture tank.

Description

technical field [0001] The invention belongs to the technical field of animal embryo engineering and relates to the establishment of a simple method for in vitro maturation and cultivation of oocytes. Background technique [0002] At present, there are two main gas-phase environments for in vitro maturation of oocytes, including 5% CO 2 +95% air (about 20% O 2 ), or 5% CO 2 +5%O 2 +90%N 2 . Studies have shown that the partial pressure of oxygen in the fallopian tubes and uterus is lower than that of air, and in hyperoxia (20% O 2 ) cultured embryos, may produce more oxygen free radicals, affecting embryonic development. When the partial pressure of oxygen is reduced from 20% to 5% to 10%, the developmental potential of the embryo will be improved. Therefore, hypoxic environment is more conducive to in vitro maturation of oocytes and embryonic development. Mingyuan will cause N in the exhaled gas of the human body 2 , O 2 , CO 2 The volume fraction was determined a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
Inventor 雷安民赵贵民吴苏君刘文强井勇杨文琳高志敏
Owner NORTHWEST A & F UNIV
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