Preparation method of heterogeneous in-vitro fertilization embryos of cows and cattle

An in vitro fertilization and cattle technology, which is applied in the field of cattle reproduction, can solve the problems of low culling rate of dairy cows, affecting in vitro fertilization, and high cost of feeding dairy cows, and achieve the effects of improving utilization rate, ensuring quality and in vitro maturation efficiency, and improving in vitro fertilization efficiency.

Active Publication Date: 2017-11-21
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, in actual production, the cost of raising dairy cows is high, and the culling rate of dairy cows is low. Even the culled cows are old, weak, and sick. Therefore, most of the cows slaughtered in slaughterhouses are old or sick cows, which seriously affects the effectiveness of in vitro fertilization. Effect

Method used

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  • Preparation method of heterogeneous in-vitro fertilization embryos of cows and cattle
  • Preparation method of heterogeneous in-vitro fertilization embryos of cows and cattle
  • Preparation method of heterogeneous in-vitro fertilization embryos of cows and cattle

Examples

Experimental program
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Effect test

Embodiment 1

[0026] [Example 1] Preparation method of Simontal cattle and yellow cattle heterogeneous in vitro fertilization embryos

[0027] (1) Put the cattle ovaries collected in the slaughterhouse into sterilized saline at 25°C with double antibodies and keep 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;

[0028] (2) The picked cumulus-oocyte complexes (COCs) were washed 3 times with DPBS buffer containing 5% fetal bovine serum (FBS) preheated in a 39°C water bath 2 hours in advance, and then washed with advance 2h in vitro maturation medium preheated in a 39°C incubator, washed 3 times and placed in CO 2 Carry out in vitro maturation culture in an incubator, using five-well plate culture, culture concentration: 100-150 pie...

Embodiment 2

[0036] [Example 2] Hoechst 33342 staining identification

[0037] (1) Take part of the oocytes fertilized in step (6) of Example 1 and wash 3 times with DPBS;

[0038] (2) 2% hyaluronidase digestion to remove cumulus cells;

[0039] (3) Hoechst 33342 dark staining for 5-7min;

[0040] (4) Observation under the fluorescence of an inverted microscope as figure 1 As shown, the left picture is the oocyte after fertilization, and the right picture shows two luminous dots under the fluorescent field of view, which are the male pronucleus and the female pronucleus stained by Hoechst 33342, which shows that the heterogeneous sperm in vitro fertilization method is feasible .

Embodiment 3

[0041] [Example 3] hatching of blastocysts

[0042] (1) Bring the collected cattle ovaries back to the laboratory in normal saline (containing double antibody) within 3 hours, and obtain cumulus-oocyte complexes (COCs) by suction;

[0043] (2) Wash the cumulus-oocyte complexes (COCs) and culture them for 22-24 hours;

[0044] (3) Simontal cattle semen is treated with BO liquid float method;

[0045] (4) the mature oocytes in step (2) are digested to remove cumulus cells;

[0046] (5) the sperm in the step (3) is moved into the fertilized fluid, and the fertilized fluid contains the oocyte in the step (4);

[0047] (6) After 6-8 hours of fertilization, culture after washing, and change the medium in half for 24 hours;

[0048] (7) After 7 days of culture, blastocysts were observed, and half of the medium was changed for 24 hours. The results were as follows: figure 2 As shown, the left picture is a blastocyst, and the right picture shows that the zona pellucida will ruptur...

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Abstract

The invention discloses a preparation method of heterogeneous in-vitro fertilization embryos of cows and cattle. The preparation method includes that cow ovaries abandoned in a slaughter house are adopted and subjected to gradient washing through normal saline in a temperature increasing type, oocytes are extracted for in-vitro maturation, and high-quality in-vitro matured oocytes are obtained; high-yield cow sperms are unfreezed and placed into a fertilization medium to be gently patted through a pipette, the cow sperms float in an incubator after overturning up and down for several times, in-vitro fertilization is performed, and the in-vitro fertilization embryos of the cows and the cattle are prepared. The oocytes are obtained through the ovaries abandoned in the slaughter house, so that utilization rate of cow resources is increased; through improvement of technical details, good effect is achieved; utilization of good male herbs can be greatly increased, the good male herbs can obtain a great deal of offspring, and function of the preparation method in genetic modification of herbs is expanded.

Description

technical field [0001] The invention belongs to the field of cattle reproduction, and in particular relates to a method for preparing in vitro fertilized embryos of cows and cattle. Background technique [0002] In vitro fertilization technology refers to the technology in which mammalian sperm and eggs complete the fertilization process in an artificially controlled environment outside the body. 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] However, in actual production, the cost of raising dairy cows is high, and the culling rate of dairy cows is l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2517/10
Inventor 张立苹林郑云郑新民毕延震华再东曹辉刘西梅肖红卫华文君李莉
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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