Preparation method of blastomere reconstructed embryo of high productive dairy cow

A blastomere, dairy cow technology, applied in the field of embryology, can solve problems such as large cell damage

Inactive Publication Date: 2018-08-21
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

The existing technology generally uses a laser membrane breaker or an oblique needle to prepare reconstructed embryos. These two methods cause relatively large damage to the cells during the preparation process.

Method used

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  • Preparation method of blastomere reconstructed embryo of high productive dairy cow
  • Preparation method of blastomere reconstructed embryo of high productive dairy cow

Examples

Experimental program
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Effect test

Embodiment 1

[0034] 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 in vitro mature dairy cow oocytes

[0035] (1) Put the dairy cow ovaries collected in the slaughterhouse into sterilized saline solution at 25°C with double antibodies and store them at a constant temperature for 7 hours, then bring them back to the laboratory, wash them with sterile saline at 30°C and 35°C in sequence, and wash them twice respectively. Put it in a water bath at 39°C for standby, and obtain cumulus-oocyte complexes (COCs) by aspiration with a 10 ml disposable syringe;

[0036] (2) The picked cumulus-oocyte complexes (COCs) were washed 3 times with DPBS buffer containing 5% fetal bovine serum (FBS) preheated in a 39°...

Embodiment 2

[0038] [Example 2] In vitro fertilization and culture to obtain high-quality dairy cow embryos

[0039] (1) Frozen semen of Simontal cattle is treated with BO liquid floatation method: the frozen semen of dairy cows is purchased from the Wuhan Dairy Cow Improvement Station, and the breed is Simontal dairy cows; the sperm processing method: take the frozen semen from a liquid nitrogen tank and put it into Thaw quickly in a 37°C water bath for 30-40 seconds, sterilize with 75% alcohol, cut the thin tube, put the sperm into a centrifuge tube containing 1mL BO solution, blow gently with a pipette gun, turn it upside down several times, and place in carbon dioxide Stand in the incubator for 20-30min to make the sperm float up, centrifuge the floated sperm at 1500r / min for 5min, then centrifuge twice with BO' solution at 1500r / min for 5min;

[0040] (2) Oocytes matured in vitro were digested with 2% hyaluronidase to remove cumulus cells, and then placed in in vitro fertilization flu...

Embodiment 3

[0045] [Example 3] Preparation of blastomere reconstituted embryos

[0046] (1) Digest the zona pellucida with 0.25% pronase to obtain a single blastomere from in vitro fertilized embryos developed to the 32-cell stage and above, and transfer them to TCM-199 operation drops containing 10% FBS for later use;

[0047] Use a fixed needle with an inner diameter of 40 μm and an outer diameter of 180 μm to suck oocytes from the direction of 9 o’clock on the hour hand, and make the first polar body in the direction of 1-3 o’clock. The operating system uses the pulse generated by its voltage effect to act on the cell surface through the injection needle, breaks the zona pellucida and cell membrane, and removes the genetic material including the first polar body. Piezo removes the first polar body parameters: break zona pellucida 3, break cell membrane 1; transfer the oocyte with the first polar body removed to the operation droplet with blastomeres for later use;

[0048] When the em...

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Abstract

The invention discloses a preparation method of a blastomere reconstructed embryo of a high productive dairy cow, and belongs to the field of embryology. The method specifically comprises the following steps: 1, preparing an in-vitro maturated dairy cow oocyte; 2, thawing frozen sperms of the high productive dairy cow; 3, preparing an in-vitro fertilized embryo, which is 32-cell stage or longer, of the high productive dairy cow; 4, constructing a blastomere reconstructed embryo of the high productive dairy cow embryo by a piezo operating system; 5, culturing the reconstructed embryo in vitro.The blastomere reconstructed embryo of the high productive dairy cow embryo is constructed by the piezo operating system, the step for preparing the reconstructed embryo by a laser system for assistedhatching or an inclined-port needle in the prior art is replaced, pulses produced by a voltage effect are directly applied to the surface of a cell for injection by the piezo operating system througha micro-injection needle, damage to the embryo is reduced greatly, and survival rate of the reconstructed embryo is increased.

Description

technical field [0001] The invention belongs to the field of embryology, and in particular relates to a method for preparing blastomere reconstituted embryos of high-yield cows. Background technique [0002] The application of in vitro fertilization method can make full use of the discarded ovaries in the slaughterhouse and the frozen semen of high-quality dairy cows for in vitro production of embryos. The production cost is reduced, and the utilization rate of the excellent breeding males is greatly increased at the same time, so that the excellent breeding males can obtain a large number of offspring, thereby expanding their role in the genetic improvement of herds. [0003] Embryo nuclear transfer technology refers to the transplantation of blastomeres and nuclei of a multicellular embryo into an enucleated oocyte or enucleated fertilized egg by micromanipulation and cell fusion to form a new embryo, and then transplanted To the recipient, biotechnology to obtain offspri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/873C12N5/073
CPCC12N5/0604C12N15/873
Inventor 张立苹林郑云郑新民毕延震刘西梅肖红卫华再东华文君曹辉
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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