Mature method for in vitro culture of porcine oocyte

A technology for in vitro culture of oocytes, applied in the field of cell biology, can solve the problems of low maturation rate, unfavorable large-scale production, long cycle of in vitro maturation of pig oocytes, etc., achieve high maturation efficiency and save manpower consumption , the effect of simple operation

Active Publication Date: 2009-05-13
深圳华大基因农业控股有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the embodiments of the present invention is to provide a method for culturing pig oocytes in vitro, which is used to solve the problem that the current technology of in vitro maturation of pig oocytes is long, Not conducive to large-scale production, the problem of low maturity rate after cultivation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] 1. Preparation of reagents and culture medium

[0077] (1) Preheating of Egg Wash (ASP):

[0078] The composition and content of egg washing liquid are:

[0079] Sodium bicarbonate 0.17mg / mL

[0080] Pyruvate 0.22mg / mL

[0081] Herpes sodium salt 3.60mg / mL

[0082] Herpes acid 2.63mg / mL

[0083] TCM-199[10x] 10% v / v

[0084] Glutamine 0.20mg / mL

[0085] Amphotericin B 3.00μg / mL

[0086] Heparin 30IU / mL

[0087] Serum 2% v / v

[0088] Preheat the egg washing solution to about 38.5°C for egg collection.

[0089] (2) Balance oocyte in vitro culture medium (IVM solution):

[0090] The composition and content of IVM liquid are:

[0091] TCM-199[1X] 80% v / v

[0092] hCG 15IU / mL

[0093] PMSG 10IU / mL

[0094] Glutamine 0.10mg / mL

[0095] Gentamicin 0.05mg / mL

[0096] Porcine follicular fluid 10%

[0097] Serum 10%

[0098] Add 400 μL of IVM solution to each well of the four-well plate to make it cover the bottom, then cover the surface of the liquid with 400 μ...

Embodiment 2

[0111] The steps of this embodiment are basically the same as in Embodiment 1, the difference is:

[0112] The composition and content of egg washing liquid are:

[0113] Sodium bicarbonate 0.17mg / mL

[0114] Pyruvate 0.22mg / mL

[0115] Herpes sodium salt 3.60mg / mL

[0116] Herpes acid 2.63mg / mL

[0117] TCM-199[10x] 5% v / v

[0118] Glutamine 0.10mg / mL

[0119] Amphotericin B 1.00 μg / mL

[0120]Heparin 10IU / mL

[0121] Serum 2% v / v

[0122] The composition and content of IVM liquid are:

[0123] TCM-199[1X] 70% v / v

[0124] hCG 5IU / mL

[0125] PMSG 6IU / mL

[0126] Glutamine 0.05mg / mL

[0127] Gentamicin 0.05mg / mL

[0128] Porcine follicular fluid 20%

[0129] Serum 5%

[0130] Results: After 36 hours of mature culture, the polar body rate was 80%-81%.

Embodiment 3

[0132] The steps of this embodiment are basically the same as in Embodiment 1, the difference is:

[0133] The composition and content of egg washing liquid are:

[0134] Sodium bicarbonate 0.17mg / mL

[0135] Pyruvate 0.25mg / mL

[0136] Herpes sodium salt 3.6mg / mL

[0137] Herpes acid 2.63mg / mL

[0138] TCM-199[10x] 15% v / v

[0139] Glutamine 0.30mg / mL

[0140] Amphotericin B 5.00μg / mL

[0141] Heparin 30IU / mL

[0142] Serum 10% v / v

[0143] The composition and content of IVM liquid are:

[0144] TCM-199[1X] 70% v / v

[0145] hCG 15IU / mL

[0146] PMSG 10IU / mL

[0147] Glutamine 0.10mg / mL

[0148] Gentamicin 0.05mg / mL

[0149] Porcine follicular fluid 12%

[0150] Serum 18%

[0151] Results: After 36 hours of mature culture, the polar body rate after culture was over 85%.

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Abstract

The invention provides a method for in vitro culture of porcine oocytes, which comprises the steps of ovary acquisition, acquisition of oocytes from an ovary, screening of the oocytes, and maturation culture. The method is characterized in that the acquired ovary is stored in a container of physiological saline for preservation before the step of the acquisition of the oocytes; and the temperature in the container is maintained to between 32 and 34 DEG C; an egg washing liquid adopted in the step of the screening of the oocytes contains amphotericin B with a concentration of between 1 and 10mu g / mL, and serum with a volume percentage of between 2 and 20 percent v / v; and the maturation medium adopted in the step of the maturation culture of the oocytes contains a TCM-199[1X] basic medium with a volume percentage of between 50 and 90 percent v / v, hGG with an antibody titer of between 5 and 25IU / mL and PMSG with an antibody titer of between 5 and 20IU / mL. The method can solve the problems that the prior in vitro maturation technology of the porcine oocytes has long period, is not good for mass production, and has low maturation rate after the culture.

Description

technical field [0001] The invention belongs to the field of cell biology, in particular to a method for culturing and maturing porcine oocytes in vitro. Background technique [0002] At present, pig embryo engineering has greatly advanced the process of animal husbandry and medicine. It can improve breeds, breed disease-resistant pigs, improve meat quality, produce transgenic pigs or genome-modified pigs, prepare human disease models, perform organ transplantation, and produce pigs. Drug proteins, research on developmental mechanisms, etc. [0003] Pig cloning and transgenic production, etc., require a large number of mature oocytes with developmental potential. So far, the in vitro maturation rate of porcine oocytes is not ideal. [0004] At present, the existing methods for culturing and maturing pig oocytes in vitro generally include the steps of collecting ovaries, collecting oocytes from the ovaries, screening oocytes and maturing oocytes. Before the oocyte collecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/075
Inventor 律波杨珍珍蔡雅兰徐颖陈玉杜玉涛
Owner 深圳华大基因农业控股有限公司
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