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Application of n-octanol to prolonging of in-vitro ageing of ovum of mammal

A mammalian and n-octanol technology, applied in the field of mammalian embryo engineering, can solve the problems of accelerating the aging of mouse eggs, achieve the effects of delaying or reversing egg aging, high innovation, and ensuring the ability of in vitro development

Inactive Publication Date: 2013-11-27
张家口桑阳牧业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, TSA (trichostatin A) can reduce the fragmentation of porcine oocytes and improve their developmental ability, but for mice, TSA can accelerate the aging of mouse eggs

Method used

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  • Application of n-octanol to prolonging of in-vitro ageing of ovum of mammal
  • Application of n-octanol to prolonging of in-vitro ageing of ovum of mammal
  • Application of n-octanol to prolonging of in-vitro ageing of ovum of mammal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Collection and in vitro maturation of bovine oocytes

[0049] Bovine ovaries were collected from the slaughterhouse, placed in a thermos flask containing saline at 25-30°C, and returned to the laboratory within 3 hours. After arriving at the laboratory, wash the ovaries with 37°C preheated sterile normal saline for 3 times, then put the washed ovaries in the preheated sterile normal saline in a 37°C water bath, and use a 10mL syringe with a 18-gauge needle Aspirate the COCs in the 2-8mm follicles above the ovary, wash cumulus cells with a complete layer of cumulus cells or part of dense cumulus cells in egg washing solution for 2 times, then wash 3 times in maturation solution, and transfer the washed COCs into Put 15-20 pieces of COCs into 100μL oocyte in vitro maturation culture micro-droplets, and finally put them in 38.5℃, 5% CO 2 and cultured in a carbon dioxide incubator with saturated humidity for 24 h.

Embodiment 2

[0050] Example 2: In vitro aging of bovine ova

[0051] After bovine COCs were cultured in vitro for 24h (0h as aging), COCs were de-cumulus cells with 0.1% hyaluronidase or COCs were transferred to TCM199+10%FBS and continued to culture under the above conditions (6-24h , put 20-25 eggs in each droplet). At different time points of aging, the naked eggs and COCs groups were randomly selected for various tests.

Embodiment 3

[0052] Example 3: Parthenogenetic Activation of Bovine Oocytes

[0053] Put the oocytes matured for 24 hours into a tube containing 0.1% hyaluronidase, repeatedly pipet with a pipette gun for 1 minute to remove cumulus cells, then wash three times with H199 solution containing 10% FBS, and remove the oocytes with complete morphology and uniform cytoplasm After the oocytes were activated in 5 μmol / L ionomycin for 5 minutes, they were transferred into 2.0 mmol / L 6-DMAP (6-dimethylaminopurine) and cultured for 4 hours. Then wash three times with CR1aa+3mg / mLBSA, transfer to CR1aa+3mg / mLBSA micro-drops pre-equilibrated for 4 hours, 15-20 pieces per drop, and place at 38.5°C, 5% CO 2 and cultured in a carbon dioxide incubator with saturated humidity, and the cleavage rate was checked 48 hours after parthenogenetic activation, and the cleaved embryos were picked out from CR1aa+3mg / mL BSA microdroplets and washed 3 times with CR1aa+10%FBS, and then Embryos were transferred to CR1aa+...

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Abstract

The invention relates to application of n-octanol to the prolonging of the in-vitro ageing of an ovum of a mammal. According to the application, the n-octanol is added in a culture solution containing fetal calf serum, and the concentration of the n-octanol is 0.1-4 mM. By using the application, the speed of the in-vitro ageing of an oocyte of the mammal is obviously deferred; meanwhile, the in-vitro development capability of the oocyte is guaranteed, so as to provide a novel approach for deferring or reversing the ageing of the ovum; and a reference is provided for improving an auxiliary reproductive technology. The technology provided by the invention is not reported in public at home and abroad, has extremely high innovativeness and has a great impellent action on the field of the embryo engineering of the mammal.

Description

technical field [0001] The invention relates to the field of mammalian embryo engineering, in particular to the application of n-octanol in delaying aging of mammalian eggs in vitro. Background technique [0002] According to whether mammalian oocytes stay in the follicle or fallopian tube for a relatively long time, they are divided into pre-ovulatory aging or post-ovulatory aging, and post-ovulatory aging can be divided into in vivo aging and post-ovulatory aging according to the environment in which the oocyte lives. In vitro aging. Oocyte aging in vitro refers to a series of biochemical and epigenetic changes that occur in mammalian eggs after a period of time in vitro. These changes lead to decreased egg fertilization rate, low fertilized embryo development and conception rate, high fetal malformation rate and postnatal mortality rate, etc. It is one of the key factors restricting the efficiency of mammalian embryo engineering. At present, there is no substantive tech...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/075
Inventor 曾申明仵毅鲍忠剑赵栓
Owner 张家口桑阳牧业有限公司
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