Serum-free bovine oocyte in-vitro maturation culture solution and oocyte culture method

A technology for in vitro maturation culture and oocyte, which is applied in the field of bovine oocyte in vitro maturation medium and oocyte culture, and can solve the problem of lack of relevant reports on the research of serum-free culture system, and achieves the improvement of the early embryo development rate and the effect of maturity

Pending Publication Date: 2020-11-17
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the in vitro maturation technology of oocytes has made great progress in recent years, there is no relevant report on the study of serum-free culture system

Method used

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  • Serum-free bovine oocyte in-vitro maturation culture solution and oocyte culture method
  • Serum-free bovine oocyte in-vitro maturation culture solution and oocyte culture method
  • Serum-free bovine oocyte in-vitro maturation culture solution and oocyte culture method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Serum-free bovine oocyte maturation medium in vitro, including Hepes-free M199 basal medium, 6mg / mL fatty acid-free BSA, 0.075IU / ml HMG, 1μg / ml 17β-estradiol, 60ng / ml EGF at 0.57mM, L-cysteine ​​at 0.57mM, bFGF at 40ng / ml, Glutamax (100x) at 1.6μL / ml, folic acid at 50μM, cholic acid at 2μg / mL and CXCL12 at 50ng / mL.

Embodiment 2

[0030] The mature culture of embodiment 2 bovine oocyte

[0031] The bovine ovaries were collected from designated slaughterhouses in Xi'an, Shaanxi Province. The ovaries were placed in a thermos flask containing 100 IU / ml penicillin and 100 μg / ml streptomycin in normal saline at 20-25°C, and transported back to the laboratory within 5 hours. After the ovary was transported back, use sterilized scissors to cut off the connective tissue, fat and attached fallopian tubes on the surface of the ovary, wash it three times in sterile normal saline, and use a 10mL syringe equipped with a 21G needle to extract the oocytes in the 2-8mm follicles on the surface of the ovary The cells were placed in a 6cm dish, and the cumulus-oocyte complexes (cumulus-oocyte complexes, COCs) were collected under a solid microscope. Wash three times in PBS after collection. COCs with normal oocyte morphology were selected for in vitro maturation culture.

[0032] Treatment group: the selected COCs were...

Embodiment 3

[0040] Embodiment 3 Preparation of bovine somatic cell cloned embryo

[0041] (1) Culture of bovine fetal fibroblasts

[0042] Take a tube of bovine fetal fibroblasts from passages 2 to 5 of Holstein cows (collected from the cattle farm of Yangling Keyuan Cloning Co., Ltd.) from liquid nitrogen and thaw at 39°C, add 0.8mL of DMEM / F12 cell culture medium, Centrifuge, discard the supernatant, add cell culture medium to resuspend, take 3mL of cell suspension and inoculate it in a 6cm-diameter petri dish, place in CO 2 Cultured at 38.5°C in an incubator.

[0043] When the bovine fetal fibroblasts reach 80% confluence, discard the culture medium and replace with Ca-free 2+ , Mg 2+ Rinse the cells with PBS, add trypsin and EDTA mixed digestion solution, and digest the cells. Observe the cells under an inverted microscope. When most of the cells retract, become round, and the intercellular space expands, stop digestion with DMEM / F12 cell culture medium containing 10% fetal bovine...

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Abstract

The invention discloses a serum-free bovine oocyte in-vitro maturation culture solution and an oocyte culture method, and belongs to the technical field of cell culture. The serum-free bovine oocyte in-vitro maturation culture solution disclosed by the invention is prepared from Hepes-free M199 basic culture solution, fatty acid-free BSA (bovine serum Albumin), HMG (high mobility group box), 17 beta-estradiol, EGF (epidermal growth factor), L-cysteine, bFGF (basic fibroblast growth factor), 100xGlutamina, folic acid, cholic acid and CXCL12. The bovine oocyte in-vitro maturation culture solution disclosed by the invention is used for culturing bovine oocytes under the condition of not using serum; the maturation rate of the bovine oocytes is improved; and the early embryo development rate of in-vitro fertilization embryos and somatic cell nuclear transplantation embryos is effectively improved.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a serum-free bovine oocyte maturation medium in vitro and a method for culturing the oocyte. Background technique [0002] In vitro maturation and culture of oocytes is the basis and key link of embryo engineering technologies such as in vitro fertilization, somatic cell nuclear transfer, in vitro production of animal embryos and transgenic cloning. In vitro maturation of oocytes is not only affected by various factors such as ovarian cycle, animal species, follicle size, hormones, and serum, but also closely related to the procedures and methods of in vitro maturation of oocytes and the components of the culture medium, especially serum will lead to in vitro production Macrosomia syndrome of embryos. Although the in vitro maturation technology of oocytes has made great progress in recent years, there is no relevant report on the study of serum-free culture system. [0003...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2500/90C12N2501/998C12N2500/30C12N2501/30C12N2501/11C12N2501/115C12N2500/38C12N2501/21
Inventor 张景程黄玥萌张敏王德宝王永张涌
Owner NORTHWEST A & F UNIV
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