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Culture solution for improving in-vitro culture quality of frozen oocytes and application of culture solution

A technology of oocytes and culture medium, applied in the field of reproduction, can solve the problem of improving frozen oocytes without cortical tension

Active Publication Date: 2022-04-15
CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the existing freezing and thawing reagents on the market are generally permeable and non-permeable cryoprotectants, which can prevent the formation of ice crystals in the freezing and thawing process by replacing the water in the oocyte to achieve the purpose of protecting the oocyte. Tension improves the quality of frozen oocytes in cryopreservation system

Method used

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  • Culture solution for improving in-vitro culture quality of frozen oocytes and application of culture solution
  • Culture solution for improving in-vitro culture quality of frozen oocytes and application of culture solution
  • Culture solution for improving in-vitro culture quality of frozen oocytes and application of culture solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1: Oocyte Collection and Freezing

[0074] 1.1 Oocyte collection

[0075] Collection of oocytes in GV stage: mice were intraperitoneally injected with 10 IU of pregnant horse serum gonadotropin, and ovary tissues of mice were collected 48 hours later. The ovaries were placed in M2 solution containing 2 μM milrinone, and the follicles were punctured with a 1 mL syringe needle under a microscope. The released GV stage oocytes were collected and placed in a 100 μL drop of M2 containing 2 μM milrinone in a 35 mm Petri dish for later use.

[0076] Oocyte collection at MII stage: mice were injected intraperitoneally with 10IU of pregnant horse serum gonadotropin, 48h after intraperitoneal injection of HCG 10IU, 12-14h after HCG injection, the mice were killed by cervical dislocation, the oviducts were taken, and the oviducts were cut and enlarged under a dissecting microscope The cumulus-oocyte complex was obtained, and the MⅡ stage oocytes discharged from the first...

Embodiment 2

[0081] Example 2 Comparative experiment of different concentrations of cortical tension enhancer ConA on the quality of GV stage frozen oocytes cultured in vitro

[0082] 2.1 In vitro maturation of frozen oocytes at GV stage

[0083] The thawed GV stage oocytes were put into M16 solutions containing different concentrations of cortical tension enhancer ConA, covered with paraffin oil and matured in vitro in an incubator.

[0084] ConA concentrations in M16 solution were: 1 μg / ml, 10 μg / ml, 100 μg / ml.

[0085] 2.2 PBE observation method

[0086] The PBE excretion rate was counted 12 hours after the GV stage oocytes matured in vitro.

[0087] 2.3 Data statistics method

[0088] All experiments were performed in at least three biological replicates, and all values ​​are presented as mean ± standard error. GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA) was used to perform unpaired two-tailed t-test on all experimental data, and P<0.05 was considered sta...

Embodiment 3

[0097] Example 3 Comparative experiment on the effect of adding ConA on the in vitro maturation and developmental ability of frozen oocytes

[0098] 3.1 Thawing the frozen oocyte of embodiment 1

[0099] When thawing, quickly take out the carrier rods to be thawed from liquid nitrogen, place them in PBS containing 0.5M sucrose for 5 minutes, and then wash them with M2 solution three times before using them for later use.

[0100] 3.2 In vitro maturation of GV stage oocytes

[0101]Divide the GV stage oocytes into three groups, namely the fresh group, the frozen group and the drug-dosed group. The fresh group is the freshly obtained GV stage oocytes, the frozen group is the thawed frozen oocytes obtained in step 3.1, and the fresh group The thawed frozen oocytes obtained in Step 3.1 were cultured in the M16 solution, and the thawed frozen oocytes obtained in step 3.1 were covered with paraffin oil in the incubator for in vitro maturation.

[0102] 3.3 MII stage oocyte thaw re...

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Abstract

The invention relates to a culture solution for improving in-vitro culture quality of frozen oocytes and application of the culture solution. According to the culture solution for improving the in-vitro culture quality of the frozen oocytes, concanavalin A is added into the M16 culture solution, and the mechanical characteristics of the frozen oocytes are recovered by adjusting cortex tension, so that the in-vitro culture quality of the frozen oocytes is improved. The invention provides a technical route for restoring the mechanical characteristics of the frozen oocytes by adjusting the cortical tension so as to improve the in-vitro culture quality of the frozen oocytes for the first time, and defines that the mechanical characteristics of the frozen oocytes can be restored by adjusting the cortical tension, the activity of spindle assembly checkpoints is enhanced, the aneuploid proportion is reduced, and the in-vitro culture quality of the frozen oocytes is improved. Further, the internal quality of in-vitro culture of the frozen oocytes is improved. According to the invention, the addition concentration of ConA in the M16 culture solution is 1-100 [mu] g / ml, preferably 10 [mu] g / ml.

Description

technical field [0001] The invention relates to the technical field of reproduction, in particular to a culture solution for improving the quality of frozen oocytes cultured in vitro and an application thereof. [0002] technical background [0003] Oocyte freezing technology is an important reproductive biology technology, which is widely used in germplasm resource preservation, fertility preservation and assisted reproductive technology. However, there are problems such as the decline of developmental potential of oocytes after thawing. It has been reported that at least 20 oocytes have been frozen. Only mature oocytes can be used to obtain offspring, which seriously restricts the wide application of this technology. Therefore, improving the utilization efficiency of frozen oocytes is an urgent problem to be solved. An in-depth analysis of the mechanism of thawing damage is the key to improving the effective utilization of frozen oocytes. premise. [0004] Oocyte developme...

Claims

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Application Information

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IPC IPC(8): C12N5/075A01N1/02
CPCY02A40/81
Inventor 傅祥伟李俊杜幸柱颛清芮侯云鹏
Owner CHINA AGRI UNIV
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