Isolated culture method of duck ovarian granulosa cells

A technology for the separation and cultivation of granulosa cells, which is applied in the field of separation and cultivation of ovarian granulosa cells, can solve the problems of unperfect and efficient methods for the separation and cultivation of duck ovary granulosa cells, and the method is simple, easy to operate, good in operability and high in vigor Effect

Inactive Publication Date: 2020-04-21
JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for the isolation and cultivation of duck ovary granulosa cells in order to overcome the problems existing in the prior art, which solves the problem that there is no perfect and efficient method for the isolation and cultivation of duck ovary granulosa cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A method for isolating and culturing duck ovary granulosa cells, the specific steps are as follows:

[0027] (1) Gaoyou duck female ducks in healthy egg-laying period (280-330 days old) were killed by bloodletting from the neck, and the abdomen feathers were wiped with 75% alcohol cotton, then the skin was cut, and the complete ovaries were taken out, and then 1% bismuth Anti-Dulbecco'sPhosphate Buffered Saline (DPBS) for washing;

[0028] (2) Place the rinsed ovary in an insulated container with 37°C 1% double antibody DPBS solution, and transfer it to the intercellular space within 4 hours;

[0029] (3) Cut the complete preovulatory follicles with high-temperature sterilized ophthalmic scissors, place each follicle in a 60mm petri dish containing 1% double-antibody DPBS solution, and use ultra-fine forceps to peel off the fine membranes and Blood vessels, rinse with 10mL syringe plus 1% double-antibody PBS solution;

[0030] (4) Cut the rinsed single preovulatory fo...

experiment example

[0037] Immunofluorescence identification of granulosa cells.

[0038] (1) Take out the duck ovary granulosa cells cultured for 48 hours, wash them with 1% double-antibody DPBS solution for 3 times, each time for 3 minutes, shake gently once at intervals, discard the double-antibody DPBS solution, and fix with 4% paraformaldehyde for 30 minutes Afterwards, pour out the paraformaldehyde, wash with 1% double anti-DPBS solution for 3 times, 3 minutes each time, keep moist and avoid light.

[0039] (2) Incubate duck ovary granulosa cells in the penetration solution (0.05% TritonX-100 and 1% Tween-20 dissolved in PBS solution) for 30 minutes at room temperature, discard the penetration solution, and use 1% double antibody DPBS solution Wash 2 times, 2min / time; add blocking solution, block at 37°C for 1h, pour off the blocking solution, and wash 3 times with 1% double antibody DPBS solution, 3min / time.

[0040] (3) Add primary antibody diluent rabbit anti-duck IgG, (primary antibody...

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PUM

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Abstract

The invention relates to the technical field of isolated culture of ovarian granulosa cells, in particular to an isolated culture method of duck ovarian granulosa cells. The isolated culture method comprises the following steps: (1) bleeding necks of female ducks to kill the female ducks, and taking out complete ovaries; (2) shearing complete follicles before ovulation to obtain single follicles before ovulation; (3) cutting off the single washed follicles before ovulation, scraping off yolk, and washing for 5 times; (4) respectively digesting cleaned granulosa cell layers by adopting hyaluronidase and trypsin; (5) resuspending the digested granulosa cell layers, conveying an obtained cell suspension into a culture medium, and repeatedly blowing and beating by using a pipettor to dispersecells; and (6) carrying out incubator culture on the resuspended cell suspension. The duck ovarian granulosa cells obtained by the isolated culture method provided by the invention are high in purityand high in activity, and the survival rate of the duck ovarian granulosa cells is high.

Description

technical field [0001] The invention relates to the technical field of separation and culture of ovary granulosa cells, in particular to a method for separation and culture of duck ovary granulosa cells. Background technique [0002] With the rapid development of large-scale intensive production of animal husbandry, the egg production of ducks is one of the most important factors restricting the development of duck industrialization, which urgently needs to be solved by breeders. The ovary is the main reproductive organ of female animals, and the periodic change of follicles on the ovary is its most important physiological activity. The graded follicles of ducks have a strict hierarchical development system, which develops and matures sequentially through primordial follicles, small white follicles, large white follicles, small yellow follicles, large yellow follicles and preovulatory follicles. In this process, ducks can adjust the speed of ovulation by regulating the numb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0682C12N2500/02C12N2509/00
Inventor 张蕾朱睿章敬旗王健张海波
Owner JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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