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Culture medium composition and culture method for promoting in-vitro development of animal embryos

A culture method and composition technology, applied in the directions of biochemical equipment and methods, embryo cells, culture process, etc., can solve the problems of inconvenient operation method, low embryo development efficiency, poor blastocyst quality, etc., and improve the in vitro development efficiency. , Simple and convenient operation, easy to operate effect

Pending Publication Date: 2021-01-15
JILIN UNIV FIRST HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Porcine SCNT embryos produced using existing SCNT techniques have low developmental efficiency and poor blastocyst quality
The existing methods to improve the efficiency of embryo development are not effective, and the operation method is not convenient enough

Method used

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  • Culture medium composition and culture method for promoting in-vitro development of animal embryos
  • Culture medium composition and culture method for promoting in-vitro development of animal embryos
  • Culture medium composition and culture method for promoting in-vitro development of animal embryos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Pig somatic cell nuclear transfer

[0047] 1. In vitro maturation culture of pig oocytes: collect freshly executed pig ovaries from a local slaughterhouse, put them in physiological saline (preheated at 35-38.5°C) containing an appropriate amount of penicillin sodium (0.768%) after collection, 2 Transport back to the laboratory as soon as possible within hours. Wash the ovaries with normal saline solution containing an appropriate amount of penicillin (0.384%), select follicles with a diameter of about 3-6 mm, and suck the follicle fluid with a sterile 10 mL syringe. Cumulus-oocyte complexes (COCs) with uniform cytoplasm and at least three layers of cumulus cells wrapped around them were selected under a stereomicroscope for in vitro maturation (IVM). Put 150-200 COCs into 1mL hormone-containing in vitro maturation medium, which is recorded as 0h, and cultured for 22-24h under the conditions of 38.5°C and 5% CO2. Then the oocytes were transferred to 1 mL hor...

Embodiment 2

[0050] Make PZM-3(-) culture drops with a volume of 60uL per drop in a 60mm Petri dish 4 hours in advance, and place at 38.5°C, 5% CO 2 Equilibrate in a carbon dioxide incubator. After the fusion of the in vitro SCNT reconstituted embryos prepared in Example 1 is completed, the 0h reconstituted embryos are dropped into the pre-balanced PZM-3(-) culture at a density of 30 per 60uL culture. Drop culture for 24h, the culture condition is 38.5℃, 5% CO 2 , and then change the medium into PZM-3-Glutamax to continue culturing until 7D. The cleavage rate was counted at 48h, and the blastocyst rate was counted at 7D.

[0051] Among them, the formula of PZM-3(-) is as follows:

[0052] Table 1

[0053]

[0054]

[0055] The formula of PZM-3-Glumax is as follows:

[0056] Table 2

[0057] 1 NaCl 108.00mM 2 KCl 10.00mM 3 KH2PO 4

Embodiment 3

[0059] Make PZM-3(-) culture drops with a volume of 60uL per drop in a 60mm Petri dish 4 hours in advance, and place at 38.5°C, 5% CO 2 Equilibrate in a carbon dioxide incubator. After the fusion of the in vitro SCNT reconstituted embryos prepared in Example 1 is completed, the 0h reconstituted embryos are dropped into the pre-balanced PZM-3(-) culture at a density of 30 per 60uL culture. Drop culture for 48h, the culture condition is 38.5 ℃, 5% CO 2 , and then change the medium into PZM-3-Glutamax to continue culturing until 7D. The cleavage rate was counted at 48h, and the blastocyst rate was counted at 7D.

[0060] Among them, the formula of PZM-3(-) is as follows:

[0061] table 3

[0062] Sodium chloride (NaCl) 106.00mM Potassium chloride (KCl) 10.05mM Potassium dihydrogen phosphate (KH2PO 4 )

0.33mM Magnesium Sulfate 7 Hydrate (MgSO 4 ·7H 2 O)

0.45mM Sodium bicarbonate (NaHCO 3 )

25.00mM Sodium pyruvate (Na-py...

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Abstract

The present invention relates to the technical field of biology and particularly to a culture medium composition and a culture method for promoting in-vitro development of animal embryos. Advantages are that the method can obviously improve in-vitro development efficiency of somatic cell nuclear transfer (SCNT) of pig embryos. An SCNT embryo blastocyst rate in a normal PZM-3 embryo culture solution is 18%-20%, the blastocyst rate of the pig SCNT can be remarkably increased to 45.7% by adopting a solution changing method, and the blastocyst rate is increased by 25.7% compared with the blastocyst rate of the normal PZM-3 embryo culture solution. Meanwhile, the method is also convenient to operate, only needs to change liquid from a PZM-3 (-) culture medium to PZM-3-Glutamax for continuous culture after the SCNT embryos are activated for 24-48 h, and is simple and convenient to operate, and suitable for subsequent application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a culture medium composition and a culture method for promoting the in vitro development of animal embryos. Background technique [0002] As early as 1962, people used somatic cell nuclear transfer technology in amphibians to reprogram terminally differentiated somatic cells to a pluripotent state. Since the first successful mammalian cloning in sheep in 1997, more than 20 cloned mammals have been obtained. Using SCNT technology can produce a complete organism from the nucleus of a single somatic cell, so this technology is used in the research of bioreactors, the production of precious medical proteins, the production of donors for xenotransplantation, the acceleration of the genetic improvement process of livestock and It has great application potential in the protection of endangered species. In addition, pluripotent embryonic stem cells can be isolated from SCNT blastocysts, wh...

Claims

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Application Information

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IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2500/12C12N2500/16C12N2500/30C12N2500/14C12N2500/33C12N2501/998C12N2500/32C12N2501/999Y02A40/70
Inventor 李子义代相鹏翟岩辉孔祥杰张胜安星兰李奇张楠
Owner JILIN UNIV FIRST HOSPITAL
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