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Method for in-vitro fertilization and embryo culture by collecting bovine oocytes in vivo

An embryo culture, oocyte technology, applied in cell culture active agents, biochemical equipment and methods, embryo cells, etc., can solve problems such as difficulty in carbon dioxide supply

Inactive Publication Date: 2020-06-12
天津力牧生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two traditional solutions. One is to put the collected oocytes in the pre-maturation solution to inhibit the maturation of the oocytes, and then transfer them to the maturation solution in the laboratory and place them in an incubator for in vitro maturation; the other is to The collected oocytes are placed in the maturation solution and transported in a micro-incubator, during which CO is continuously supplied. 2 , but it is more difficult to maintain the supply of carbon dioxide in the transport state

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the culture method of bovine in vitro fertilization embryo (ex vivo collection)

[0049] 1. Reagents

[0050] In the specific test of the present invention, if not otherwise specified, the relevant reagents used are described in detail as follows:

[0051] Add double-antibody normal saline: normal saline containing 400 IU / mL penicillin and 400 μg / mL streptomycin.

[0052] Egg washing solution: BY basal culture solution supplemented with 3mg / mL bovine serum albumin.

[0053] Maturation medium: BY basal medium supplemented with 100 mL / L FBS, 10 μg / mL FSH, 10 μg / mL LH, 1 μg / mL E2, and 20 ng / mL EGF.

[0054] Mature medium containing HEPES: BY basal medium supplemented with 15mmol / L HEPES, 100mL / L FBS, 10μg / mL FSH, 10μg / mL LH, 1μg / mL E2, 20ng / mL EGF.

[0055] Among them, EGF—epidermal growth factor, FSH—follicle stimulating hormone, FBS—fetal bovine serum, E2—estradiol, LH—luteinizing hormone.

[0056] Fertilization medium: containing 112.0mM sodium chlo...

Embodiment 2

[0089] Embodiment 2: the culture method of bovine in vitro fertilization embryo (living body collection)

[0090] 1. Reagents

[0091] In the specific test of the present invention, if not otherwise specified, the relevant reagents used are described in detail as follows:

[0092] Add double-antibody normal saline: normal saline containing 400 IU / mL penicillin and 400 μg / mL streptomycin.

[0093] Egg washing solution: BY basal culture solution supplemented with 3mg / mL bovine serum albumin.

[0094] Maturation medium: BY basal medium supplemented with 100 mL / L FBS, 10 μg / mL FSH, 10 μg / mL LH, 1 μg / mL E2, and 20 ng / mL EGF.

[0095] Mature medium containing HEPES: BY basal medium supplemented with 15mmol / L HEPES, 100mL / L FBS, 10μg / mL FSH, 10μg / mL LH, 1μg / mL E2, 20ng / mL EGF.

[0096] Among them, EGF—epidermal growth factor, FSH—follicle stimulating hormone, FBS—fetal bovine serum, E2—estradiol, LH—luteinizing hormone.

[0097] Fertilization medium: containing 112.0mM sodium...

Embodiment 12

[0130] Embodiment 12: Culture method of bovine in vitro fertilization embryo (living body collection)

[0131] The main difference between this example and the above example 2 is that in step (1), the collected oocytes are placed in the transport medium containing HEPES at 38.8°C, without carbon dioxide supply, and transported back to the laboratory within 24 hours.

[0132] 1. Reagents

[0133] In the specific test of the present invention, if not otherwise specified, the relevant reagents used are described in detail as follows:

[0134]The transport culture fluid used in this example contains: glycine 50.0mg / L, L-alanine 25.0mg / L, L-arginine hydrochloride 70.0mg / L, L-aspartic acid 30.0mg / L L, L-cystine dihydrochloride 26.0mg / L, L-glutamic acid 75.0mg / L, L-glutamine 100.0mg / L, L-histidine hydrochloride monohydrate 21.88mg / L L, L-hydroxyproline 10.0mg / L, L-isoleucine 40.0mg / L, L-leucine 60.0mg / L, L-lysine hydrochloride 70.0mg / L, L-formazine Thionine 15.0mg / L, L-phenyla...

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Abstract

The invention relates to a method for in-vitro fertilization and embryo culture by collecting bovine oocytes in vivo. Specifically, on one hand, the bovine in-vitro fertilization embryo culture methodcomprises the following steps of: taking follicular fluid derived from ovum pick-up of bovine living body, picking out a cumulus-oocyte complex wrapped with at least containing three layers of cumulus cells under a stereoscopic microscope, putting the cumulus-oocyte complex into a transport culture solution, and transporting the cumulus-oocyte complex back to a laboratory within 24 hours; washingCOCs once in an oocyte maturation culture solution and then transferring into a new maturation culture solution for culturing for 22-24 h under the culture conditions that the temperature is 38.8 DEGC, the concentration of CO2 is 5.5-6.5% and the humidity is saturated; performing in vitro fertilization; and carrying out embryo in vitro culture and preservation. The invention also relates to a transport culture solution for oocytes. The method and the transportation culture solution disclosed by the invention show excellent technical effects as shown in the specification.

Description

technical field [0001] The invention belongs to the technical field of animal reproduction, and relates to a technology for agricultural-animal husbandry and veterinary reproduction, in particular to a method for culturing bovine in vitro fertilized embryos. In addition, the present invention also relates to the use of related culture fluids for the cultivation of bovine in vitro fertilized embryos. Application in in vitro fertilization embryo culture. Further, the present invention also relates to a method for culturing bovine IVF embryos using the relevant culture fluid for culturing bovine IVF embryos. In particular, the method for culturing bovine IVF embryos of the present invention has excellent technical effects. In particular, the present invention relates to a method for live collective oocytes from cattle for in vitro fertilization and embryo culture. Background technique [0002] In Vitro Fertilization (In Vitro Fertilization) or (external fertilization) refers ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073A61D19/04A01N1/02
CPCA01N1/0284A61D19/04C12N5/0604C12N2500/32C12N2500/38C12N2500/40C12N2501/11C12N2501/31C12N2501/998
Inventor 王娜王小武赵明礼郝少强郭晶张月桥郭春明
Owner 天津力牧生物科技有限公司
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