Method for enhancing ectogenesis of sheep oocyte

A technology of oocyte and in vitro development, applied in artificial cell constructs, germ cells, animal cells, etc., which can solve the developmental ability of few sheep oocytes

Active Publication Date: 2012-08-01
INNER MONGOLIA SAINUO GRASSLAND SHEEP IND
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned existing methods are used in cattle, mice, and pigs, but there are few reports on improving the developmental ability of sheep oocytes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0023] Pick up sheep ovaries that were slaughtered within 30 minutes, put them in physiological saline containing 1000IU / ml penicillin and 1000IU / ml streptomycin at a temperature of 30°C, wash 4 times with normal saline within 3 hours; 100 μMol / L FSK and 500 μMol / L IBMX egg extraction fluid, a 10ml syringe with a 9-gauge needle to aspirate follicles with a size of 6 mm on the surface of the ovary, and the recovered follicle fluid in the syringe was placed in a 60 mm dish and examined under a microscope Pick out the oocytes, place them in the egg extraction solution for 2 hours, wash 3 times with the in vitro maturation solution containing 20 μMol / L CIL, and place them in the CO 2 Incubate in the box for 2 hours, CO 2 The meteorological condition of the box is 5% CO 2 , 95% air, the temperature is 38.6 ℃. Then wash 3 times with CIL-free in vitro maturation solution, CO 2 Cultivate in the box for 24 hours, wash 4 times with in vitro fertilization fluid, add 30 pieces / drop int...

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Abstract

The invention provides a method for enhancing the ectogenesis of sheep oocyte. The method adopts an adenylate cyclase activator Forskolin (FSK), phosphodiesterase inhibitor cilostamide (CIL) and 3-isobutyl-1-methylxanthine (IBMX), treats the sheep oocyte in a combining manner and improves the capability of the ectogenesis of the sheep oocyte. The method comprises the following detailed steps of: picking the ovary of the killed sheep, putting the ovary of the killed sheep into normal saline for cleaning for 3-4 times; pumping the 2-8mm follicles on the surface of the ovary by using a 10ml syringe which absorbs 1ml ovum-pumping liquid containing 100mu mol / L FSK and 500mumol / L IBMX and is provided with a number-9 needle head, and injecting the follicular fluid recovered in the syringe into a35-60mm vessel; picking the oocyte under a microscope, putting the oocyte into the pumped follicular fluid, cleaning the oocyte for three times with in-vitro mature fluid containing 20mu mol / L CIL, and putting the oocyte in a CO2 incubator for culture; then cleaning the oocyte for three times with in-vitro mature fluid which does not contain CIL, putting the oocyte in the CO2 incubator for culture and adding into in-vitro fertilization fluid drop; unfreezing the seminal fluid with water bathing, and commonly incubating with the oocyte; and calculating the cleavage rate after 24 hours and calculating the blastocyst rate after 168 hours.

Description

technical field [0001] The present invention relates to promoting the in vitro development of sheep oocytes, using adenylate cyclase activator Forskolin (FSK), phosphodiesterase inhibitor cilostamide (CIL) and 3-isobutyl-1-methylxanthine 3 -isobutyl-1-methylxanthine (IBMX), combined with treatment of sheep oocytes, can significantly improve their in vitro developmental ability. Background technique [0002] The use of in vitro fertilization technology in animal husbandry can produce test-tube embryos in batches in a factory form, thereby providing more high-quality embryos for livestock embryo transfer and promoting the improvement of livestock breeding. In vitro fertilization technology mainly includes three links: in vitro maturation of oocytes, sperm capacitation and in vitro culture of embryos. [0003] For mammalian oocytes, high concentrations of cyclic adenosine monophosphate (cAMP) can inhibit meiosis, thereby inhibiting the nuclear maturation of oocytes, creating t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075C12N5/071
Inventor 黄俊成汪立芹林嘉鹏赵云程唐森玛依拉
Owner INNER MONGOLIA SAINUO GRASSLAND SHEEP IND
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