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Method for cloning embryo with cattle spermatozoon

A technology for cloning embryos and sperm, applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, to achieve the effect of reducing the requirements of operating skills and increasing accuracy

Inactive Publication Date: 2011-04-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From this point of view, although there may be theoretical challenges in sperm cloning, they can all be overcome, and the theory on small animals may not necessarily apply to large animals

Method used

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  • Method for cloning embryo with cattle spermatozoon
  • Method for cloning embryo with cattle spermatozoon
  • Method for cloning embryo with cattle spermatozoon

Examples

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Effect test

Embodiment 1

[0055] 1. Sampling: Store the ovaries of healthy yellow cattle cows collected from the Jiangnan Cattle Slaughterhouse in Wuhan City, Hubei Province in phosphate buffered solution at 39°C (see "Contents of the Invention" for the formula), and bring them back to the laboratory within 3 hours;

[0056] 2. Collection and cultivation of bovine oocytes: absorb bovine cumulus cell-oocyte complexes from bovine ovary surface 2-8 mm follicles with a syringe (commercially purchased veterinary needle No. 16), and the bovine cumulus cells- The oocyte complex was washed 3 times with the maturation culture solution (see the content of the invention for the formula), and then transferred to 100 μl of the maturation culture solution (see the content of the invention for the formula) and cultured in microdrops for 22 hours (the culture conditions were: 39°C, 100% relative humidity, 5% CO 2 incubator);

[0057] 3. Judgment of the maturity of bovine oocytes: the bovine oocytes cultured in the ma...

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Abstract

The invention relates to a new method for cloning embryo with bovine sperms, which comprises the steps as follows: (1) a collected bovine ovary is cleaned, and an ovarium mound-oocyte complex is drawn out for in vitro culture to lead the oocyte to be cultured and developed to a second meiosis metaphase; (2) then dyeing and fluorescence localization are carried out on the nucleolus of the oocyte,and the nucleolus is denucleated to obtain the denucleated bovine oocyte; (3) the semen of an excellent breeding bull is collected or the commercial semen of a self-sex controlled excellent breeding bull is selected and stored in a liquid nitrogen container; (4) the semen is unfrozen and capacitation treatment is carried out on the semen; then the semen is arranged in the injecta of the oocyte; (5) two capacitation sperms are injected into the cytoplast of the denucleated bovine oocyte to obtain a sperm reconstructed embryo; (6) the sperm reconstructed embryo is activated and cultured. The invention overcomes the single mode that the donor cell in the traditional nuclear transfer can only be a somatocyte, and is a new method for generating a bovine embryo by using double sperms to replacethe somatocyte as a donor to be injected into the cytoplast of denucleated bovine oocyte.

Description

technical field [0001] The invention relates to the technical field of mammalian reproduction, in particular to a new method for cloning embryos using bovine sperm. Background technique [0002] Since the successful use of somatic cell nuclear transfer technology to obtain surviving cloned sheep [1], various mammals and even some primate mammals have been cloned successfully. Despite the low efficiency of animal cloning techniques [2], it has been well demonstrated that oocyte cytoplasm and donor nuclei can support the entire embryonic development process. And somatic cell nuclear transfer technology has revolutionary application prospects in regenerative medicine, new drug development, animal breeding, etc. Therefore, animal cloning technology is still a research hotspot at home and abroad. [0003] However, there are relatively few studies on animal germ cell cloning at home and abroad. And considering that the primordial germ cells are in the state of elimination of gen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/06
Inventor 杨利国王滇肖遥张鑫霍立军
Owner HUAZHONG AGRI UNIV
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