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Single-semen injection activation method

A fertilized egg and 6-DMAP technology, which is applied in the field of embryo engineering, can solve the problems such as the decline of embryo development ability

Inactive Publication Date: 2008-09-10
BEIJING JINXIU DADI AGRI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The combined treatment of ethanol and cycloheximide can increase the activation rate, and the effect is significantly better than the single treatment of ethanol or cycloheximide (Presiece, 1994). With the increase of egg age, the activation rate of ethanol increases, but the egg age is partial High (maturity culture 44h) its embryonic development ability declines (Minamihashi, 1993)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Embodiment 1 (joint processing)

[0012] 1 reagent

[0013] 1.1M199 (Medium 199 GIBCO 11150-059)

[0014] 1.2 Glucose (D-Glucose SIGMA G-8270)

[0015] 1.3 Caffeine (Caffeine SIGMA C-0750)

[0016] 1.4 Fetal bovine serum (FBS GIBCO 26140-087)

[0017] 1.5 Hyaluronic acid enzyme (H SIGMA H4272)

[0018] 1.6 Cytochalasin B (CB SIGMA C6762)

[0019] 1.7 Dimethyl sulfoxide (DMSO SIGMA D4540)

[0020] 1.8io (ionomycin SIGMA I0634)

[0021] 1.96-DMAP (6-Dimethylaminopurine SIGMA D2629)

[0022] 1.10DDT (GIBCO D1532)

[0023] 1.11PVP (SIGMA P5288)

[0024] 2 Experimental equipment and instruments

[0025] 2.1CO 2 Incubator Model3111

[0026] 2.2 Solid Microscope M-400

[0027] 2.3 Biological Microscope XSZ-G

[0028] 2.4 Centrifuge JAI SHI LI XIN JI80-2B

[0029] 2.5 incubator DHP120

[0030] 2.6 Constant temperature water bath Q / 1YS002-91 BS

[0031] 3 operation steps

[0032] 3.1 Preparation of oocytes The oocytes obtained from the isolated ovary or live oo...

Embodiment 2

[0043] Embodiment 2 (ionomycin treatment)

[0044] The steps are as in Example 1, but step 3.4 is different

[0045] 3.4 Oocyte activation

[0046] 3.4.1 Transfer the fertilized eggs into the solution with a final concentration of 5 μMio for 6 minutes.

[0047] 3.4. After 26 minutes, fully wash with the culture medium for 3-5 times, put it into the culture drop, and put it into the culture medium (M199+10% FBS) in the incubator for cultivation.

Embodiment 3

[0048] Embodiment 3 (DMAP processing)

[0049] The steps are as in Example 1, but different under step 3.4

[0050] 3.4 Oocyte activation

[0051] 3.4.1 Transfer fertilized eggs into 6-DAMP with a final concentration of 2 μM, wash 3 times with 6-DAMP solution, put them into prepared droplets, and place them in an incubator for 3 hours.

[0052] 3.4. After 23 hours, the fertilized eggs were taken out, fully washed with culture medium for 3-5 times, put into culture drops, and cultured in culture medium (M199+10% FBS) in an incubator.

[0053] Result statistics: record of embryonic development

[0054] Handling of reconstructed embryos

[0055] The above table was statistically analyzed by t test. It can be seen that the cleavage rate and blastocyst rate of Example 1 are the best among the three methods, and the cleavage rate is significantly different from the other two methods (P<0.05).

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PUM

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Abstract

The invention relates to an intracytoplasmic sperm injection activation method, which belongs to the embryo engineering field. The invention uses ionomycin and DMAP to activate a fertilized ovum together, thereby the activation rate of oocytes and the cleavage rate are improved; after the ionomycin with a final concentration of 5 mu m and the DMAP with a final concentration of 2 mu m are used for activating the fertilized ovum, the cleavage rate is 60 to 65 percent.

Description

technical field [0001] The invention relates to the field of embryo engineering, in particular to the technique of producing in vitro embryos activated by single sperm injection. Background technique [0002] Single sperm injection is the process of artificially injecting a single sperm into the egg cytoplasm to fertilize it, form a pronucleus, and finally develop into an early embryo in vitro. The method of injecting a single sperm directly into the bovine oocyte cytoplasm to complete the fertilization process is called intracytoplasmic injection. In order to achieve high-efficiency in vitro production of embryos of fine breeds of cattle, the method of single sperm intracytoplasmic injection has become the preferred method of sexual reproduction in the world. In developed countries, this method has replaced the traditional method of in vitro fertilization and has become the industrial production The main technology of embryos. In our country, this technology has made grea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06A61B17/43C12N15/873
Inventor 王海张红霞郭敏王月安晶郭丽丽张家新
Owner BEIJING JINXIU DADI AGRI CO LTD
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