CN104982419B - Buffalo embryo freezing method in combination with laser membrane rupture - Google Patents
Buffalo embryo freezing method in combination with laser membrane rupture Download PDFInfo
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- CN104982419B CN104982419B CN201510454904.6A CN201510454904A CN104982419B CN 104982419 B CN104982419 B CN 104982419B CN 201510454904 A CN201510454904 A CN 201510454904A CN 104982419 B CN104982419 B CN 104982419B
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Abstract
The present invention provides a buffalo embryo freezing method in combination with laser membrane rupture. The method comprises: culturing an in vitro fertilized buffalo embryo to be an expanded blastocyst; using a laser membrane rupture instrument to drill a blastocyst cavity; discharging blastocyst fluid; and then using freezing liquid to perform vitrification freezing. According to the present invention, discharging the blastocyst fluid before freezing can make the cryoprotectant enter an embryo more quickly, reduce the possibility of ice crystal forming in a cell, and thereby avoiding embryo damage to a large extent, and improving a freezing survival rate of the embryo. On the other hand, due to partial breaking of a zona pellucida before freezing, so that after thawing cultivation, the zona pellucida is easier to be hatched in a revived blastocyst cavity, and thereby facilitating embryo implantation of the embryo after transplanting, and improving a transplant pregnancy rate.
Description
[technical field]
The invention belongs to biological technical field, particularly to the buffalo embryo freezing method of a kind of combination laser rupture of membranes.
[background technology]
Babalus bubalis L. produced in vitro embryo is low to low-temperature sensitive and freezing efficiency, causes embryo's matter mainly due to condition of in vitro culture is improper
Measure the best and embryo's lactone to drip and cause more.For the cryotherapy of buffalo embryo, mainly use vitrifying freeze process,
But for the standard that embryo cryopreservation stage, freezing liquid and freezing procedure are the most unified.And buffalo embryo before freezing the most not
Through any process, therefore freezing efficiency is the most relatively low, and for the blastaea of 8-9d, its freezing efficiency is only 50-60%.
Additionally, due to after glass freezing-defrosting, the zona pellucida thickness of embryo averagely can increase by 2.8 μm, the embryonic hatching after freezing
It is also only about 50%, thus causes the conception rate after defrosting embryo transfer to be only 4-12%.It is thus desirable to a kind of freezing hands of development
Section ensures the freezing efficiency of buffalo embryo, improves incubation rate and the Pregnancy rate of embryo after freeze-thaw simultaneously.
[summary of the invention]
The present invention provides the buffalo embryo freezing method of a kind of combination laser rupture of membranes, and freeze survival rate and the transplanting that can improve embryo are subject to
Tire rate.
The technical solution used in the present invention is:
The buffalo embryo freezing method of a kind of combination laser rupture of membranes, described method is to expansion by the embryo culture of Babalus bubalis L. produced in vitro
Blastaea, punches segmentation cavity with laser rupture of membranes instrument, discharges blastaea liquid, then carries out glass freezing with freezing liquid.
Further, described freezing liquid is made up of following material: concentration is ethylene glycol (EG) solution of 20%, and concentration is 20%
Dimethyl sulfoxide (DMSO) solution, concentration is the sucrose solution of 0.5M.
Further, said method comprising the steps of:
(1) XYClone laser rupture of membranes system is used, adjustment parameter to 90% pulse strength, the pulse width of 800 μ s,
Make the zona pellucida otch of 15-20 μm on the zona pellucida on blastaea inner cell mass opposite, and break up segmentation cavity and make blastaea liquid stream go out；
(2) take 0.25mL plastics straw and make the oblique section of one 30 ° at its one end blade, as buffalo embryo cryotherapy
Carrier；
(3) keep 5min in the embryo handled well is proceeded to the freezing balance liquid after equilibrium at room temperature, then proceed in freezing liquid
Clean 2-3 time, draw the blastaea a small amount of freezing liquid of mixing (less than 1 μ L) and be positioned over the tip of straw oblique section, direct plunge into Liquid Nitrogen
Preserve；
(4) in liquid nitrogen container, take out the embryo of Ultra-cryofreezing preservation, be directly immersed in the thawing solution of equilibrium at room temperature, rock wheat
Pipe, is shown in that blastaea is deviate under the microscope, and blastaea stops 5min in thawing solution, then with CM liquid (embryo medium)
Clean 2-3 time, proceed in the 40-50 μ micro-liquid of L CM of covering paraffin oil, 38.5 DEG C, 5%CO2And under conditions of saturated humidity
Cultivation makes it recover.
Further, described freezing liquid, freezing balance liquid, thawing solution all use TCM199 and 10% hyclone (FBS) to mix
Close liquid based on solution.
Further, described freezing balance liquid is made up of following material: concentration is the EG solution of 20%, and concentration is the DMSO of 5%
Solution.
Further, described thawing solution is made up of following material: concentration is the sucrose solution of 0.5M.
The invention has the beneficial effects as follows:
Blastaea liquid is discharged before freezing by the present invention, frozen solution can be made to enter embryo faster, and decrease intracellular ice
The brilliant probability formed, thus largely avoid embryo damage, the freeze survival rate of embryo can be improved.On the other hand,
Abolish owing to just zona pellucida to have been carried out part before freezing, therefore after cultivation of thawing, the segmentation cavity of recovery can be made to be more easy to hatch
Zona pellucida, thus beneficially implantation in uterus after embryo transfer, improve Pregnancy rate.
[detailed description of the invention]
Further illustrate the present invention below in conjunction with specific embodiment, but do not limit the scope of the present invention with this.
Embodiment 1
The buffalo embryo freezing method of a kind of combination laser rupture of membranes, described method is to expansion by the embryo culture of Babalus bubalis L. produced in vitro
Blastaea, punches segmentation cavity with laser rupture of membranes instrument, discharges blastaea liquid, then carries out glass freezing with freezing liquid.In this enforcement
In mode, described freezing liquid is made up of following material: concentration is ethylene glycol (EG) solution of 20%, and concentration is the diformazan of 20%
Base sulfoxide (DMSO) solution, concentration is the sucrose solution of 0.5M.Further, the Babalus bubalis L. embryo of described combination laser rupture of membranes
Tire freezing method is further comprising the steps of:
(1) using XYClone laser rupture of membranes system, adjustment parameter is to 90% pulse strength, and the pulse width of 800 μ s, at capsule
Make the zona pellucida otch of 15-20 μm on the zona pellucida on the inner cell mass opposite of embryo, and break up segmentation cavity and make blastaea liquid stream go out.
(2) take 0.25mL plastics straw and make the oblique section of one 30 ° at its one end blade, as buffalo embryo ultra-low temperature cold
Freeze carrier.
(3) keep 5min in the embryo handled well is proceeded to the freezing balance liquid after equilibrium at room temperature, then proceed in freezing liquid
After cleaning, drawing the blastaea a small amount of freezing liquid of mixing (less than 1 μ L) and be positioned over the tip of straw oblique section, direct plunge into Liquid Nitrogen is protected
Deposit.In the present embodiment, described freezing balance liquid, freezing liquid all use TCM199+10% hyclone (FBS) with thawing solution
Liquid based on mixed liquor；Described freezing balance liquid is made up of following material: concentration is the EG solution of 20%, and concentration is 5%
DMSO solution.
(4) in liquid nitrogen container, take out the embryo of Ultra-cryofreezing preservation, be directly immersed in the thawing solution of equilibrium at room temperature, rock straw,
See that blastaea is deviate under the microscope.Blastaea stops 5min in thawing solution, then cleans with CM liquid (embryo medium)
2-3 time, proceed in the 40-50 μ L CM microdroplet of covering paraffin oil, 38.5 DEG C, 5%CO2And cultivation makes under conditions of saturated humidity
Its recovery.In the present embodiment, described thawing solution is made up of following material: concentration is the sucrose solution of 0.5M.
The blastaea of matched group processes without rupture of membranes, directly carries out freezing according to above-mentioned method, and rupture of membranes group is i.e. to be carried out by blastaea
Freezing is carried out again after rupture of membranes.Experimental result is as follows:
The impact on not homeomorphism frozen-thawed blastocysts in age survival rate of table 1. rupture of membranes
Note: data of going together in table relatively go up table difference lowercase alphabet and show significant difference (P < 0.05).
Table 2. rupture of membranes is on the impact of incubation rate after not homeomorphism frozen-thawed blastocysts in age
Note: data of going together in table relatively go up table difference lowercase alphabet and show significant difference (P < 0.05).
After laser rupture of membranes, the freeze survival rate of the freezing blastaea to embryo being 6-7d age and the incubation rate after thawing do not have as seen from the table
Have a significant impact, but improve 8d blastaea freeze survival rate and thaw after incubation rate, especially significantly improve 9d blastaea
Freeze survival rate and incubation rate.
The two of post-thaw survival groups of blastaeas are implanted into respectively the receptor water cow intrauterine through Estrus synchronization, result table
The 6d blastaea Pregnancy rate of bright matched group and rupture of membranes group is respectively 31.7% (13/41) and 32.9% (24/73), 7d capsule
Embryonic implantation conception rate is respectively 35.7% (5/14) and 22.9% (8/35), and two groups are all not significantly different from.And transplant 8-9d
Conception rate after blastaea is respectively 4.2% (1/24) and 18.2% (4/22).As can be seen here, the present invention can significantly improve
The Pregnancy rate of 8-9d freezing blastaea.
Viewed from above, the present invention has good freezing efficiency, incubation rate and Pregnancy rate for freezing 6-7d blastaea,
The freeze survival rate of 8-9d blastaea, incubation rate and Pregnancy rate can be improved especially, thus be greatly improved buffalo embryo
Utilization ratio, the genetic improvement for Babalus bubalis L. has great importance.
Claims (5)
1. the buffalo embryo freezing method combining laser rupture of membranes, it is characterized in that: described method is by the embryo culture of Babalus bubalis L. produced in vitro to Blastocysts, is punched by segmentation cavity with laser rupture of membranes instrument, discharges blastaea liquid, then carry out glass freezing with freezing liquid, comprise the following steps:
(1) use XYClone laser rupture of membranes system, adjust parameter to 90% pulse strength, the pulse width of 800 μ s, the zona pellucida on blastaea inner cell mass opposite makees the zona pellucida otch of 15-20 μm, and break up segmentation cavity and make blastaea liquid stream go out；
(2) take 0.25mL plastics straw and make the oblique section of one 30 ° at its one end blade, as buffalo embryo cryotherapy carrier；
(3) keeping 5min in the embryo handled well proceeds to the freezing balance liquid after equilibrium at room temperature, clean 2-3 time in then proceeding to freezing liquid, draw blastaea and mix with the freezing liquid less than 1 μ L, be positioned over the tip of straw oblique section, direct plunge into Liquid Nitrogen preserves；(4) in liquid nitrogen container, take out the embryo of Ultra-cryofreezing preservation, it is directly immersed in the thawing solution of equilibrium at room temperature, rock straw, see that blastaea is deviate under the microscope, blastaea stops 5min in thawing solution, then clean 2-3 time with embryo medium, proceed in the 40-50 μ micro-liquid of L CM of covering paraffin oil, 38.5 DEG C, 5%CO2And cultivation makes it recover under conditions of saturated humidity.
The buffalo embryo freezing method of combination laser rupture of membranes the most according to claim 1, it is characterized in that: described freezing liquid is made up of following material: concentration is ethylene glycol (EG) solution of 20%, concentration is dimethyl sulfoxide (DMSO) solution of 20%, and concentration is the sucrose solution of 0.5M.
The buffalo embryo freezing method of combination laser rupture of membranes the most according to claim 1, it is characterised in that: described freezing liquid, freezing balance liquid, thawing solution all use liquid based on TCM199 and 10% hyclone (FBS) mixed solution.
The buffalo embryo freezing method of combination laser rupture of membranes the most according to claim 1, it is characterised in that: described freezing balance liquid is made up of following material: concentration is the EG solution of 20%, and concentration is the DMSO solution of 5%.
The buffalo embryo freezing method of combination laser rupture of membranes the most according to claim 1, it is characterised in that: described thawing solution is made up of following material: concentration is the sucrose solution of 0.5M.
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| CN109644991A (en) * | 2019-01-31 | 2019-04-19 | 力盟低温医学(深圳)有限公司 | In conjunction with freezing sample processing method of the laser technology under without permeability protective agent |
| CN110122477B (en) * | 2019-06-13 | 2021-07-06 | 台州恩泽医疗中心(集团) | Anti-stress cryopreservation and unfreezing method for mouse embryos thinned by combining zona pellucida |
| CN112825849B (en) * | 2021-03-22 | 2024-02-13 | 上海理工大学 | Modularized high-flux low-temperature preservation and laser rewarming device |
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| JP2525713B2 (en) * | 1993-01-19 | 1996-08-21 | 農林水産省畜産試験場長 | Culture and transport method of bovine embryo using low molecular weight thiol compound |
| AU2002334378B2 (en) * | 2001-08-23 | 2008-02-28 | Reliance Life Sciences Pvt.,Ltd | Isolation of inner cell mass for the establishment of human embryonic stem cell (hESC) lines |
| CN101003791A (en) * | 2007-01-11 | 2007-07-25 | 山东省农业科学院畜牧兽医研究所 | Refrigeration method for preserving embryo and oocyte by vitrification of glasscapillary |
| CN102771473A (en) * | 2012-04-25 | 2012-11-14 | 余文莉 | Freezing liquid for preserving embryo, preparation method and application thereof |
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| CN104488853B (en) * | 2015-01-12 | 2016-08-24 | 中国农业大学 | A kind of ox embryo vitrifying freeze gets rid of pipe and thaws and directly transplanting method |
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Effective date of registration: 20221024 Address after: Room 406, Floor 4, Production Workshop, No. 65, Fengda Road, Nanning Hi tech Industrial Development Zone, 530000 Guangxi Zhuang Autonomous Region Patentee after: Huangshi Saier Biotechnology (Guangxi) Co.,Ltd. Address before: 530001 no.24-1 Yongwu Road, Xingning District, Nanning City, Guangxi Zhuang Autonomous Region Patentee before: GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INSTITUTE |