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Oocyte separation and extraction method based on duck embryo

An oocyte and extraction method technology, applied in the field of oocyte separation and extraction, can solve the problems of affecting research, time-consuming, affecting oocyte extraction, etc., so as to reduce cell contamination, reduce test costs, and achieve fast and efficient acquisition. Effect

Inactive Publication Date: 2017-09-15
ANIMAL SCI RES INST GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method does not extract a single oocyte, which affects subsequent research, and is not suitable for poultry whose follicle structure is different from that of mammals, because poultry follicles are not only smaller than livestock follicles, but also have a large number of follicles. Yolk, affecting oocyte extraction
Existing methods generally select adult livestock to separate and extract oocytes, which takes a long time

Method used

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  • Oocyte separation and extraction method based on duck embryo
  • Oocyte separation and extraction method based on duck embryo
  • Oocyte separation and extraction method based on duck embryo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A method for separating and extracting oocytes based on duck embryos, comprising the following steps:

[0032] Step 1, select 4 ovary tissues of duck embryos at 25 embryonic age, place the ovary tissues in sterilized PBS, and cut the ovary tissues into pieces with ophthalmic scissors;

[0033] Step 2. Add mixed enzyme digestion solution to the shredded ovarian tissue fragments (each 10ml digestion solution contains 5 mg of collagenase II, protease, and hyaluronidase each), and place the ovarian tissue fragments and mixed enzyme digestion solution in a fine mouth In the digestion bottle, digest on a shaker for 45min, 37°C, 190r / min;

[0034] Step 3. Blow and beat the ovarian tissue fragments with a pipette gun, add 10% fetal bovine serum to stop the digestion, and filter through a 200-mesh cell sieve;

[0035] Step 4. Collect the filtrate and centrifuge for 3.5 minutes at a rotational speed of 1200r / min; discard the filtrate, add cell culture medium to the precipitated ...

Embodiment 2

[0038] A method for separating and extracting oocytes based on duck embryos, comprising the following steps:

[0039] Step 1. Select 6 ovarian tissues of duck embryos with an embryonic age of 24 days, place the ovarian tissues in sterilized PBS, and cut the ovarian tissues into pieces with ophthalmic scissors;

[0040] Step 2. Add mixed enzyme digestion solution (each 10ml digestion solution contains collagenase II, protease, and hyaluronidase 4mg) to the shredded ovarian tissue fragments, and place the ovarian tissue fragments and mixed enzyme digestion solution in a fine mouth In the digestion bottle, digest on a shaker for 50min, 37°C, 170r / min;

[0041] Step 3. Blow and beat the ovarian tissue fragments with a pipette gun, add 10% fetal bovine serum to stop the digestion, and filter through a 200-mesh cell sieve;

[0042] Step 4. Collect the filtrate and centrifuge for 3 minutes at a rotational speed of 1500r / min; discard the filtrate, add cell culture medium to the preci...

Embodiment 3

[0045] A method for separating and extracting oocytes based on duck embryos, comprising the following steps:

[0046] Step 1. Select 5 ovarian tissues of duck embryos with an embryonic age of 26 days, place the ovarian tissues in sterilized PBS, and cut the ovarian tissues into pieces with ophthalmic scissors;

[0047]Step 2. Add mixed enzyme digestion solution (each 10ml digestion solution contains collagenase II, protease, and hyaluronidase 3 mg) to the shredded ovarian tissue fragments, and place the ovarian tissue fragments and mixed enzyme digestion solution in a fine mouth In the digestion bottle, digest on a shaker for 60min, 37°C, 180r / min;

[0048] Step 3. Blow and beat the ovarian tissue fragments with a pipette gun, add 10% fetal bovine serum to stop the digestion, and filter through a 200-mesh cell sieve;

[0049] Step 4. Collect the filtrate and centrifuge for 4 minutes at a rotational speed of 1000r / min; discard the filtrate, add cell culture medium to the preci...

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Abstract

The invention discloses an oocyte separation and extraction method based on a duck embryo. The method comprises the following steps: selecting ovary tissue of a duck embryo, putting the ovary tissue into a sterilized PBS (Phosphate Buffer), and cutting the ovary tissue; adding a mixed enzyme digestive solution into the cut ovary tissue, putting ovary tissue fragments and the mixed enzyme digestive solution into a small-mouth digestive bottle, and performing digestion treatment on a shaking table; beating the ovary tissue fragments by using a pipette, adding fetal calf serum to terminate digestion, and filtering by using a cell screening net; collecting filtrate, and performing centrifugal treatment; abandoning the filtrate, adding a cell culture medium into precipitated cells, gently beating by using the pipette to suspend the cells, and laying a cell suspension on a culture dish; performing separation and extraction, so as to obtain oocyte based on the duck embryo. By adopting the method, grown livestock does not need to be dissected, cell contamination is reduced, the testing cost is reduced, and the oocyte can be rapidly and efficiently acquired.

Description

technical field [0001] The invention belongs to the technical field of poultry breeding, and in particular relates to a method for separating and extracting oocytes based on duck embryos. Background technique [0002] At present, in vitro maturation of oocytes in domestic animals is a reproductive biotechnology. The oocytes derived from ovarian antral follicles can provide abundant egg sources for breeding, cloning and transgenic animals through in vitro maturation, and this technology has been produced is widely applied. The most important thing in this technology is to isolate a large number of oocytes with good activity. In this regard, livestock research has been very mature, but poultry has rarely been reported. [0003] In the current research report, the in vitro culture of livestock oocytes is obtained from preantral follicles by collagenase digestion to obtain a large number of oocyte-granulosa cell complexes derived from 0.2-0.3mm preantral follicles. However, th...

Claims

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Application Information

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IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2509/00
Inventor 罗茜夏伟光郑春田陈伟何静怡董春琳
Owner ANIMAL SCI RES INST GUANGDONG ACADEMY OF AGRI SCI
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