Micro fertilization method

A technology of micro-fertilization and fertilized eggs, applied in the direction of vertebrate cells, artificial cell constructs, animal cells, etc., can solve the problems of complicated operation, unfavorable production promotion and high cost of micro-fertilization technology, and achieve the convenience of large-scale production and promotion , cost reduction, and ease of operation

Inactive Publication Date: 2012-05-16
陈浩杰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the shortcomings of micro-fertilization technology are co

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 sperm preparation

[0034] 1. Fresh sperm:

[0035] Take rabbits (New Zealand rabbits aged 6-8 months, SPF grade, provided by Shanghai Slack Experimental Animal Co., Ltd., license number: SCXK2004-0005) fresh semen with D-PBS solution (commercially purchased) 2000r / min Centrifuge and wash three times, then slowly add D-PBS solution, and place at 38.5°C, 5% CO 2 , in an incubator at 100% humidity for 10 minutes. The frozen-thawed semen of rabbits was directly put into D-PBS solution, and upstream under the same conditions for 10 minutes.

[0036] 2. Freeze-thawed sperm:

[0037] Collect rabbit semen and freezing liquid (the formula of freezing liquid is: add 3.52g Tris HCL, 1.96g citric acid, and 1.59g glucose into 100ml deionized water and mix well as the base liquid; 100ml freezing liquid: add 20ml egg yolk, 4ml Glycerin, mix and pack, store in refrigerator at 4°C.) Dilute and mix at 1:1, slowly cool down at 4°C, seal with 0.25-0.30ml plastic thin tube ...

Embodiment 2

[0038] Example 2 Oocyte preparation

[0039] 1. Fresh Oocytes

[0040] Donor female rabbits were primiparous New Zealand rabbits aged 6-8 months and weighing about 3kg. According to the method of Kennelly and Foote (1965), 2 times a day (12h interval) for 3 consecutive days, each subcutaneous injection of FSH50IU / only (Ningbo 12h after the last injection of FSH, inject 100 IU of HCG (manufactured by Ningbo Second Hormone Factory) into the ear vein and mate with ligated male rabbits. Oocyte retrieval was carried out 14, 16, and 18 hours after HCG injection. Cut off the fallopian tubes under sterile conditions, absorb 10ml of oviposit fluid, flush the eggs from the bell mouth to the junction of the uterine tube, use 5mL oviposit fluid on each side, put the cumulus oocyte complex in a container containing 0.2% In the Eppendorf tube of hyaluronidase D-PBS solution, shake and digest on a vortex mixer for 5 minutes to remove granule cells, pick out the enzyme-digested oocytes und...

Embodiment 3

[0047] Example 3 injecting sperm into oocytes

[0048] 1. Manufacture of operation panel

[0049] The operation plate is designed according to the style introduced in the third edition of the ICSI chapter of >, that is, 4-5 rows of operation droplets are sequentially made on the cover of the 90mm cell culture dish, and each row of operation solution The drop consists of three 3ul 10% PVP (polyvinylpyrrolidone) drops and three 5ul mTCM199 drops, absorb 2ul of floating upper layer sperm into the second PVP drop, put the eggs in the fourth mTCM199 drop, and the rest in the middle of the dish The small drops are used for cleaning the holding tube and the injection needle. After all the operation drops are completed, 3-5ml of paraffin oil is used to cover the entire operation panel. The design style of the operation panel operation drop is shown in the figure ( figure 1 ).

[0050] 2. Addition of spermatozoa in PVP drops

[0051] Add PVP (1gPVP poured into 10ml D-PBS solution)...

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Abstract

The invention provides a micro fertilization method, which mainly comprises the following steps of: 1) taking fresh seminal fluid or frozen thawed seminal fluid; 2) taking fresh oocyte cells after the human chorionic gonadotropin (HCG) is injected for 14 to 18 hours; 3) respectively injecting the sperm in the fresh seminal fluid and the live sperm in the frozen thawed seminal fluid into the oocyte cells obtained in the second step to obtain injection oocyte; 4) activating the injection oocyte by using 5mumol/L ionomycin and 2mmol/L 6-dimethyaminopurine (6-DMAP) to obtain fertilized ovum; and 5) placing the fertilized ovum into culture liquid consisting of TCM199, 10 percent fetal bovine serum (FBS), 1.25 mM sodium pyruvate and 0.1 mM ethylene diamine tetraacetic acid (EDTA) to be cultured. The oocyte cell cytoplasm introcytoplasmic sperm injection method provided by the invention is simple and has the advantages that the operation is easy, and in addition, the large-scale production and popularization is convenient.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an in vitro culture technique, in particular to a microscopic fertilization method. Background technique [0002] Microfertilization is widely used in basic reproductive research and human assisted reproductive technology to treat infertility. Historically, in mammals, microfertilization first started in golden hamsters; since then, microfertilization has provided invaluable information for the study of mammalian fertilization mechanisms. Due to the advancement of animal genetic engineering and reproductive technology, microfertilization technology has been widely used to identify the biological significance of specific genes or to confirm the normal form of genes in gametes after experimental manipulation in vitro. [0003] Oocyte intracytoplasmic sperm injection (ICSI), as a means of assisted microfertilization, has developed rapidly in just a few decades. In terms of the nature and...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 陈浩杰
Owner 陈浩杰
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