Culture solution and method for improving in-vitro maturation quality of porcine oocytes

A technology for in vitro maturation of oocytes, applied in the direction of cell culture active agents, animal cells, tissue culture, etc., to achieve the effects of promoting development rate and embryo quality, solving poor quality, and improving maturation rate and oocyte quality

Pending Publication Date: 2019-12-31
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still no research showing whether Asiatic acid can improve the quality of oocytes cultured in vitro. Therefore, adding Asiatic acid to the in vitro culture medium can improve the quality of oocytes, which is beneficial to the production of high-quality oocytes and early embryonic development. has important practical significance

Method used

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  • Culture solution and method for improving in-vitro maturation quality of porcine oocytes
  • Culture solution and method for improving in-vitro maturation quality of porcine oocytes
  • Culture solution and method for improving in-vitro maturation quality of porcine oocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the preparation of the in vitro maturation culture fluid containing subacid

[0030] A culture solution for improving the quality of porcine oocyte maturation in vitro, said culture solution comprising the following components: M199 culture solution, 10ng / mL EGF, inosinic acid, 0.6mM L-cysteine, 0.91mM sodium pyruvate, 10% follicular fluid, 10IU / mL LH, 10IU / mL FSH, and 100IU / mL penicillin.

[0031] The M199 culture solution, EGF, Asiatic acid, L-cysteine, sodium pyruvate, and penicillin were all purchased from Sigma Company, and the LH and FSH were purchased from Ningbo Second Hormone Factory. The product number of Asiatic acid is 546712.

[0032] The culture solution preparation process comprises the following steps:

[0033] (1) Weigh 97.74 mg of Asiatic acid, dissolve it in 2 mL of DMSO and filter it with a 0.22 μm filter to prepare a 100 mM concentrated stock solution of Asiatic acid;

[0034] (2) The culture solution described in claim 1 is made int...

Embodiment 2

[0039] Embodiment 2: the mature culture of porcine oocyte

[0040] A culture method for improving the quality of in vitro maturation of pig oocytes, comprising the following steps:

[0041] (1) Collect freshly slaughtered pig ovaries from the slaughterhouse, put them in 37°C physiological saline with double antibodies, and send them to the laboratory within 2 hours; put the culture medium prepared in the above step (4) into 38.5 °C, 5% CO 2 , 95% air, and a carbon dioxide incubator with saturated humidity for 3 hours in advance as the experimental group. In the control group, 10 μM Asiatic acid was replaced with DMSO of the same concentration, and other experimental conditions were the same;

[0042] (2) Wash the ovaries with 0.9% normal saline at 37°C for 2-3 times, temporarily store the washed ovaries in a water bath at 37°C, select a 10mL syringe with a 12-gauge needle, and press the ovaries by hand Generate a certain negative pressure, and extract 3-8mm follicles from on...

Embodiment 3

[0054] Example 3: Application of Asiatic Acid to Improving the Maturation Quality of Pig Oocytes in Vitro

[0055] 1. Parthenogenetic activation of oocytes

[0056] Parthenogenetic activation proceeds as follows:

[0057] (1) Preparation of decumulus cells: Use a 200 µL pipette to suck out the oocytes of the experimental group and the control group in Example 2 from the culture medium, put them into 0.1% hyaluronidase, and pipette Gently blow with the gun, observe under the microscope until all the cumulus cells fall off, and pick them out with a straw;

[0058] (2) Washing: wash the decumulus cells with HEPES buffer for 2-3 times, wash away the granulosa cells, and pick out the dead oocytes at the same time;

[0059] (3) Select oocytes: transfer the decumulus cells washed in step (2) from the microdroplets of HEPES buffer solution to the microdroplets of operating solution and wash them for 2-3 times, and the first polar body will be discharged in good shape. The oocytes w...

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Abstract

The invention discloses a culture solution and method for improving the in-vitro maturation quality of porcine oocytes. The culture solution is prepared from the following components: an M199 culturesolution, 10 ng / mL of EGF, 10 [mu]M of asiatic acid, 0.6 mM of L-cysteine, 0.91 mM of sodium pyruvate, 10% follicular fluid, 10 IU / mL of LH, 10 IU / mL of FSH and 100 IU / mL of penicillin. According to the culture solution and method, the asiatic acid is added into the porcine oocyte in-vitro maturation culture solution for the first time, the maturation rate of the oocytes can be effectively increased, the quality of the oocytes can be effectively improved, the development rate of the oocytes can be effectively increased, the quality of the oocytes can be effectively improved, the developmentalrate of parthenogenetic embryos and in-vitro fertilized embryos can be increased, the embryo quality of the parthenogenetic embryos and the in-vitro fertilized embryos can be improved, and the problems of low efficiency and poor quality of in-vitro maturation and in-vitro culture at present are effectively solved.

Description

technical field [0001] The invention relates to a culture solution and a culture method for improving the quality of pig oocytes, in particular to a culture solution and a culture method for improving the quality of in vitro maturation of pig oocytes, and belongs to the application field of animal reproduction technology. Background technique [0002] With the rapid development of animal embryo engineering, the requirements for the quality and quantity of oocytes are gradually increasing. Due to the limitation of the number of mature oocytes in vivo and the methods of obtaining them, the in vitro maturation of oocytes has become an important source of embryo production in vitro and plays an important role in the field of animal reproduction. Therefore, in vitro maturation of oocytes recovered from slaughterhouses is the most economical and effective means. The quality of oocytes is of great significance for fertilization, early embryo development and embryo transfer. How to...

Claims

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Application Information

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IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2501/11C12N2500/30C12N2500/32C12N2501/31
Inventor 梁爽孙博兴张嘉保齐佳佳李晓霞王大力胡玮宜
Owner JILIN UNIV
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