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Method for culturing single porcine embryo in vitro

A culture method and embryo body technology are applied in the field of in vitro culture of pig parthenogenetically activated embryos and single embryos to achieve the effect of simple preparation

Active Publication Date: 2018-10-26
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to different species, embryo size, embryo in vitro culture conditions (including culture medium, culture temperature, etc.) single embryo in vitro culture

Method used

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  • Method for culturing single porcine embryo in vitro
  • Method for culturing single porcine embryo in vitro
  • Method for culturing single porcine embryo in vitro

Examples

Experimental program
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Effect test

Embodiment 1

[0044] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The examples provided are only illustrative of the method of the present invention and do not limit the rest of the present disclosure in any way. [Example 1] Preparation of porcine in vitro matured oocytes

[0045] After the sow ovaries were collected in Wuhan COFCO Meat Food Processing Plant, they were immediately placed in sterilized saline at 35°C with double antibodies and sent back to the laboratory within 2-4 hours. The ovarian tissue was first washed with 75% alcohol for 1 min, then washed with sterilized and preheated saline for 3 times, and then the surface diameter of the ovary was extracted with a sterile syringe equipped with a standard 20-gauge needle (with a small amount of balanced DPBS inside). For follicles of 3-8mm, the extraction solution was poured into a 50mL conical centrifuge tub...

Embodiment 2

[0047] [Example 2] Parthenogenetic activation of porcine in vitro matured oocytes

[0048] Transfer the COCs matured in vitro for 42-44 hours into DPBS containing 0.1% hyaluronidase for digestion, gently blow repeatedly with a pipette gun to remove cumulus cells, wash 3 times with pre-heated activation solution, and place in a In the fusion tank of the liquid, the BTX-2001 fusion instrument is used for electrical activation, and the activation parameters are: 30μs, 1.1kv / cm, 1 direct current pulse. The activated oocytes were washed three times with preheated embryo culture medium NCSU-23 before use.

[0049] Activation solution formula: 0.25mol / L mannitol+0.5mol / L calcium chloride+0.5mol / L magnesium sulfate+0.5mol / LHEPES+0.01%PVA.

Embodiment 3

[0050] [Example 3] Preparation of microdroplets of co-cultured feeder cells

[0051] (1) Preparation of fallopian tube epithelial cell feeder layer microdroplets

[0052] A. Isolation and primary culture of fallopian tube epithelial cells: Obtain the fallopian tubes of sows from the slaughterhouse, put them in 30-37°C sterilized saline with double antibodies, bring them back to the laboratory within 2 hours, and cut off the excess with sterilized scissors Mesosalpinx, and cut the fallopian tubes into 2cm-long sections, wash them with sterile saline at 35°C for 3 times, place them in a disposable plastic dish with a diameter of 35mm, and wash out the epithelium while observing it under a stereomicroscope When operating, the left hand holds the tweezers of a clock to clamp the fracture on one side of the fallopian tube, and the right hand uses a 1 ml disposable syringe to absorb the working concentration of 0.25% trypsin and directly inject it slowly from the fracture of the fal...

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Abstract

The invention discloses a method for culturing a single porcine embryo in vitro, and belongs to the field of agriculture-animal husbandry and veterinary medicine. The method particularly involves thatfeeder layer cell micro-droplets are adopted to be co-cultured with a single embryo so as to obtain a better development result; the development process of single embryo culture is consistent with the development process of traditionally used multi-embryo culture, and the single embryo attached with a transparent belt or removing a transparent belt can obtain a porcine parthenogenetic developmentblastosphere in 7 days. The method for culturing the single porcine embryo in vitro has the advantages that the provided feeder layer cell micro-droplets preparation is simple, a reliable original material for studying the development of a single embryo can be provided according to the provided method, and the needs of tracking, observing and further researching the development process of the single porcine embryo can be fully met; the provided single embryo culture method can be widely applied to single embryo culture of various species.

Description

technical field [0001] The invention belongs to the field of agriculture-animal husbandry and veterinary medicine, and in particular relates to a single embryo in vitro culture method of pig parthenogenetic activated embryos. Background technique [0002] In recent years, in vitro production of embryos has been widely used in the production of economically valuable animals, disease models, and bioreactors. At the same time, in vitro fertilization (IVF), single sperm injection (ICSI), embryo transfer, and embryo segmentation The application of embryonic stem cell engineering has greatly promoted the development of animal husbandry. However, in the development and application of these new technologies, the low developmental ability of embryos produced in vitro and the developmental arrest of early embryos have greatly limited these new technologies. Technology development and application. Although many studies have improved the developmental ability of embryos by adding or re...

Claims

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Application Information

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IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2500/30C12N2500/34
Inventor 张立苹林郑云毕延震华再东郑新民
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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