CN110122477B - Anti-stress cryopreservation and unfreezing method for mouse embryos thinned by combining zona pellucida - Google Patents
Anti-stress cryopreservation and unfreezing method for mouse embryos thinned by combining zona pellucida Download PDFInfo
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses an anti-stress cryopreservation and unfreezing method for a mouse embryo thinned by combining with a zona pellucida, and belongs to the technical field of embryo preservation. The anti-stress cryopreservation method comprises the following steps: (1) preparing an anti-stress refrigerating fluid; (2) thinning the embryo zona pellucida; (3) and (3) putting the thinned embryo into a freezing balance liquid for balancing, transferring the embryo into a freezing liquid to clean and transfer among different liquid drops when the embryo is recovered to 80% of the original volume, and putting the embryo onto a carrying rod within 1 minute and quickly immersing the embryo into liquid nitrogen. The invention can obviously improve the embryo freezing recovery rate, protect the development potential of the frozen embryo and reduce the abortion rate after pregnancy by thinning the zona pellucida and matching with the use of the anti-stress refrigerating fluid.
Description
Technical Field
The invention relates to the technical field of embryo preservation, in particular to a stress-resistant cryopreservation and thawing method for a mouse embryo thinned by combining a zona pellucida.
Background
The embryo freezing preservation technology is one of the key technologies of embryo transplantation, solves the problem that the embryo transplantation is limited by time and place, and brings the superiority of the embryo transplantation into full play. At present, the mouse embryo freezing mainly comprises slow freezing and vitrification freezing, but the two methods have certain defects. The slow freezing body needs an expensive programmed freezing instrument, the in vitro balance operation time of the embryo is long, the freezing operation is complex, the in vitro retention time of the embryo is long, and the embryo is badly stressed, and in addition, the recovery rate of the method is 70-80 percent, and the recovery rate is low. The vitrification freezing adopts the high-concentration cryoprotectant, so that the embryo freezing time is shortened, the freezing recovery rate is higher, but the toxicity and the stress injury of the high-concentration cryoprotectant to the embryo in-vitro operation exist. Therefore, the influence of the in vitro embryo freezing operation time and the stress factors of the in vitro operation environment on the subsequent development of the embryo needs to be further changed, so as to achieve the freezing method which not only improves the freezing preservation effect, but also does not influence the further development potential of the embryo.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a stress-resistant cryopreservation and thawing method for a mouse embryo combined with a thinned zona pellucida.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a stress-resistant cryopreservation method for a mouse embryo thinned by combining a zona pellucida comprises the following steps:
(1) preparation of anti-stress refrigerating fluid: the anti-stress refrigerating fluid comprises a freezing equilibrium fluid and a refrigerating fluid;
the frozen equilibrium liquid adopts HTF as a basic liquid, and 7-8% by volume of EG, 7-8% by volume of DMSO, 11-13% by volume of HSA, 150-250um/L vitamin C and 90-100um/L vitamin E are added;
HTF is adopted as a base liquid of the refrigerating fluid, 12-16% by volume of EG, 13-16% by volume of DMSO, 0.5M sucrose, 10-15% by volume of HSA, 150-250um/L vitamin C and 90-100um/L vitamin E are added;
(2) thinning of embryo zona pellucida: thinning the zona pellucida of the embryo at the cleavage stage of the third day after fertilization in a mouse, taking the embryo at the 6-10 cell stage of the third day, marking the embryo by using a ruler, dividing the embryo into four regions on average, thinning the zona pellucida of one region in the middle of the zona pellucida by using a laser film-breaking instrument, wherein the total thinned region accounts for one fourth of the embryo, the pulse width of the laser is 0.200ms, the aperture is 2-3um, thinning is carried out by using continuous laser, the zona pellucida is thinned for one fourth in three times, the pulse track is adjusted to be matched with the radian of the zona pellucida during each thinning, 6 pulse laser thinning is carried out continuously each time, and finally, the zona pellucida is cut at two ends of the thinned region;
(3) putting the thinned embryo into a freezing balance liquid for balancing, transferring the embryo into a freezing liquid to clean and transfer among different liquid drops when the embryo is restored to 75-85% of the original volume, putting the embryo on a carrying rod within 1 minute, and quickly immersing the embryo into liquid nitrogen.
Preferably, in the step (1), HTF is used as a base liquid, 7.5% by volume of EG, 7.5% by volume of DMSO, 12% by volume of HSA, 200um/L of vitamin C and 100um/L of vitamin E are added;
HTF is adopted as a base liquid of the refrigerating fluid, 15% by volume of EG, 15% by volume of DMSO, 0.5M sucrose, 12% by volume of HSA, 200um/L vitamin C and 100um/L vitamin E are added.
Further, the specific operation steps of the step (3) are as follows:
s1, placing the embryo on the surface of the first frozen equilibrium liquid drop, allowing the embryo to freely fall to the bottom of the first frozen equilibrium liquid drop, and staying in the first frozen equilibrium liquid drop for 2 min; then transferring the embryo to the bottom of the second frozen equilibrium liquid drop and purging for 3 times, and staying in the second frozen equilibrium liquid drop for 1 min; then transferring the embryo to the bottom of the third frozen equilibrium liquid drop and blowing for 3 times, and observing the condition of the blastomere of the embryo;
and S2, when the embryo is recovered to 80% of the original volume, transferring the embryo into the freezing liquid drop, sucking the embryo to the bottom of the first freezing liquid drop, allowing the embryo to float to the upper part of the liquid surface, then quickly transferring the embryo to the bottom of the second freezing liquid drop for purging for 3 times, then quickly transferring the embryo to the carrying rod after again transferring to the bottom of the third freezing liquid drop for purging for 3 times, and placing the embryo into liquid nitrogen for freezing and storing.
Further, in step S2, the retention time of the embryo in the freezing fluid is controlled within 60S.
The invention also provides a thawing method of the frozen mouse embryo, which comprises the following steps: taking the frozen mouse embryo, quickly inserting a carrying rod with the embryo into TS1 liquid preheated to 37 ℃ for balancing for 1min, transferring the embryo into TS2 liquid for balancing for 3min, transferring the embryo into TS3 liquid for balancing for 5min, finally balancing for 5min in WS liquid, transferring the embryo into a culture medium, putting the culture medium into a culture box, evaluating the survival condition of the embryo after culturing for 2 hours, and then continuously culturing the surviving embryo until the blastocyst stage is incubated;
the TS1 liquid, the TS2 liquid, the TS3 liquid and the WS liquid are all prepared by taking HTF as base liquid, wherein 1M sucrose, 200um/L vitamin C and 12% HSA in volume percentage are added into the TS1 liquid; 0.5M sucrose, 200um/L vitamin C and 12% HSA by volume are added into the TS2 liquid; 0.25M sucrose, 200um/L vitamin C and 12% HSA by volume are added into the TS3 liquid; WS solution was supplemented with 200um/L vitamin C and 12% by volume HSA.
Further, the specific thawing operation steps of the mouse embryo are as follows:
A. inserting the tail end of the carrying rod with the embryo into TS1 liquid preheated to 37 ℃, separating the embryo from the carrying rod by using a suction pipe, and staying in TS1 liquid for 1 min;
B. adding the embryo with TS1 liquid to the surface of the first TS2 liquid drop to allow the embryo to freely fall down and stay for 1min, then transferring the embryo to the bottom of the second TS2 liquid drop and blowing and sucking for 3 times and staying for 1min, and then transferring the embryo to the bottom of the third TS2 liquid drop and blowing and sucking for 3 times and staying for 1 min;
C. transferring the embryo with the TS2 liquid to the surface of a first TS3 liquid drop, allowing the embryo to fall freely and stay in the liquid drop for 1min, then transferring the embryo to the bottom of a second TS3 liquid drop and sucking for 3 times and staying for 2min, and then transferring the embryo to the bottom of a third TS3 liquid drop and sucking for 3 times and staying for 2 min;
D. transferring the embryo to the bottom of the first WS droplet for 3 times, transferring the embryo to the second WS droplet for 2min, transferring to the bottom of the third WS droplet for 3 times, staying for 3min, and transferring to a blastocyst culture medium for continuous culture.
The invention adopts the method of cleaning and transferring the embryo among different liquid drops to operate in the steps of freezing and storing, the transfer among the liquid drops is mainly carried out for keeping stability, the balance and the environment of freezing liquid are more suitable for the freezing and the unfreezing of the mouse embryo, and the development potential of the unfrozen embryo can be kept in a better state.
Has the advantages that:
the invention can lead the cryoprotectant to rapidly enter the embryo for balancing by thinning the zona pellucida, shorten the embryo freezing balancing time, reduce the adverse effect of the environment, and simultaneously the thinned zona pellucida is beneficial to the incubation after the embryo recovers, thereby improving the embryo implantation and pregnancy rate; by improving the formula of the refrigerating fluid and adding vitamin E and vitamin C, the damage of active oxygen generated by in vitro operation can be effectively prevented, the development potential of the frozen embryo is protected, and the abortion rate after pregnancy is reduced.
Drawings
FIG. 1 is a diagram of embryos marked with rulers and divided into regions;
FIG. 2 is a diagram of an embryo laser thinned starting from the middle of a zona pellucida;
FIG. 3 is a diagram of a thinned embryo in the region of zona pellucida 1/4;
FIG. 4 is a diagram of an embryo with zona pellucida severed at either end of the thinned region.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
Example 1
A stress-resistant cryopreservation method for a mouse embryo thinned by combining a zona pellucida comprises the following steps:
(1) preparation of anti-stress refrigerating fluid: the anti-stress refrigerating fluid comprises a freezing equilibrium fluid and a refrigerating fluid;
the frozen equilibrium liquid adopts HTF as a base liquid, and 7.5 percent by volume of EG, 7.5 percent by volume of DMSO, 12 percent by volume of HSA, 200um/L vitamin C and 100um/L vitamin E are added;
HTF is adopted as a base liquid of the refrigerating fluid, 15% by volume of EG, 15% by volume of DMSO, 0.5M sucrose, 12% by volume of HSA, 200um/L vitamin C and 100um/L vitamin E are added.
(2) Thinning of embryo zona pellucida:
frozen embryo acquisition on the third day after fertilization of mice:
experimental materials: female ICR mice at 4-6 weeks, male mice at about 12 weeks;
superovulation and cage combination of mice:
A. first day 23 pm: 00 female mice were injected with PMSG 10IU (0.1 ml);
B. afternoon on the third day 23: 00 the female mice are injected with hCG 10IU (0.1ml) and mated with the male mice;
C. the fourth day, 9 am: 00 female mice were tested for vaginal emboli to determine if mating was successful.
Collecting mouse embryos in a cell stage and freezing the embryos to obtain:
2 cell murine embryos were removed 40 hours after hCG injection. Spraying 75% alcohol on mouse abdomen skin, cutting skin from the lower side of abdomen to V shape, cutting peritoneum, finding uterus and ovary on two sides of peritoneum, cutting uterine horn and oviduct between ovaries, putting into culture dish containing MHTF, inserting embryo acquisition device into umbrella of oviduct, washing out 2-cell embryo, collecting embryo into washing culture dish, washing, transferring into culture dish, culturing in CO2 culture box for the third day, and selecting 6-10 cell embryo for freezing.
Thinning embryo zona pellucida at the cleavage stage of the third day after fertilization in the body of the mouse:
taking embryos in 6-10 cell stages on the third day, marking the embryos by using a ruler and dividing the embryos into four areas on average as shown in figure 1, thinning the zona pellucida in one area by using a laser membrane-breaking instrument as shown in figure 2-3, wherein the total thinned area accounts for one fourth of the embryos, the laser pulse width is 0.200ms, the aperture is 2-3um, thinning is carried out by using continuous laser, the zona pellucida is thinned for one fourth in three times, the pulse track is adjusted to be consistent with the radian of the zona pellucida during each thinning, 6 pulse laser thinning is continuously carried out each time, and finally, the zona pellucida is cut at two ends of the thinned area as shown in figure 4.
(3) And (3) putting the thinned embryo into a freezing balance liquid for balancing, transferring the embryo into a freezing liquid to clean and transfer among different liquid drops when the embryo is recovered to 80% of the original volume, and putting the embryo onto a carrying rod within 1 minute and quickly immersing the embryo into liquid nitrogen. The specific operation steps are as follows:
s1, placing the thinned embryo on the surface of the first frozen equilibrium liquid drop, allowing the embryo to freely fall to the bottom of the first frozen equilibrium liquid drop, and staying in the first frozen equilibrium liquid drop for 2 min; then transferring the embryo to the bottom of the second frozen equilibrium liquid drop and purging for 3 times, and staying in the second frozen equilibrium liquid drop for 1 min; then transferring the embryo to the bottom of the third frozen equilibrium liquid drop and blowing for 3 times, and observing the condition of the blastomere of the embryo;
and S2, when the embryo is recovered to 80% of the original volume, transferring the embryo into the freezing liquid drop, sucking the embryo to the bottom of the first freezing liquid drop, allowing the embryo to float to the upper part of the liquid surface, then quickly transferring the embryo to the bottom of the second freezing liquid drop for purging for 3 times, then transferring the embryo to the bottom of the third freezing liquid drop for purging for 3 times, controlling the retention time of the embryo in the freezing liquid within 60S, quickly transferring the embryo to a carrying rod, and placing the carrying rod into liquid nitrogen for freezing and storing.
The thawing of the frozen mouse embryo includes the following steps: taking the frozen mouse embryo, quickly inserting a carrying rod with the embryo into TS1 liquid preheated to 37 ℃ for balancing for 1min, transferring the embryo into TS2 liquid for balancing for 3min, transferring the embryo into TS3 liquid for balancing for 5min, finally balancing for 5min in WS liquid, transferring the embryo into a culture medium, putting the culture medium into a culture box, evaluating the survival condition of the embryo after culturing for 2 hours, and then continuously culturing the surviving embryo until the blastocyst stage is incubated; the specific thawing operation steps are as follows:
A. inserting the tail end of the carrying rod with the embryo into TS1 liquid preheated to 37 ℃, separating the embryo from the carrying rod by using a suction pipe, and staying in TS1 liquid for 1 min;
B. adding the embryo with TS1 liquid to the surface of the first TS2 liquid drop to allow the embryo to freely fall down and stay for 1min, then transferring the embryo to the bottom of the second TS2 liquid drop and blowing and sucking for 3 times and staying for 1min, and then transferring the embryo to the bottom of the third TS2 liquid drop and blowing and sucking for 3 times and staying for 1 min;
C. transferring the embryo with the TS2 liquid to the surface of a first TS3 liquid drop, allowing the embryo to fall freely and stay in the liquid drop for 1min, then transferring the embryo to the bottom of a second TS3 liquid drop and sucking for 3 times and staying for 2min, and then transferring the embryo to the bottom of a third TS3 liquid drop and sucking for 3 times and staying for 2 min;
D. transferring the embryo to the bottom of the first WS droplet for 3 times, transferring the embryo to the second WS droplet for 2min, transferring to the bottom of the third WS droplet for 3 times, staying for 3min, and transferring to a blastocyst culture medium for continuous culture.
The TS1 liquid, the TS2 liquid, the TS3 liquid and the WS liquid are all prepared by taking HTF as base liquid, wherein 1M sucrose, 200um/L vitamin C and 12% HSA in volume percentage are added into the TS1 liquid; 0.5M sucrose, 200um/L vitamin C and 12% HSA by volume are added into the TS2 liquid; 0.25M sucrose, 200um/L vitamin C and 12% HSA by volume are added into the TS3 liquid; WS solution was supplemented with 200um/L vitamin C and 12% by volume HSA.
Example 2
A stress-resistant cryopreservation method for a mouse embryo thinned by combining a zona pellucida comprises the following steps:
(1) preparation of anti-stress refrigerating fluid: the anti-stress refrigerating fluid comprises a freezing equilibrium fluid and a refrigerating fluid;
the frozen equilibrium liquid adopts HTF as a base liquid, and 8% by volume of EG, 7% by volume of DMSO, 11% by volume of HSA, 150um/L vitamin C and 100um/L vitamin E are added;
HTF is adopted as a base liquid of the refrigerating fluid, 16% by volume of EG, 13% by volume of DMSO, 0.5M sucrose, 15% by volume of HSA, 150um/L vitamin C and 100um/L vitamin E are added;
(2) thinning of embryo zona pellucida: the same as example 1;
(3) putting the thinned embryo into a freezing balance liquid for balancing, transferring the embryo into a freezing liquid to clean and transfer among different liquid drops when the embryo is restored to 85 percent of the original volume, and putting the embryo on a carrying rod within 1 minute and quickly immersing the embryo into liquid nitrogen; the procedure was as in example 1.
The thawing of the frozen mouse embryo includes the following steps: taking the frozen mouse embryo, quickly inserting a carrying rod with the embryo into TS1 liquid preheated to 37 ℃ for balancing for 1min, transferring the embryo into TS2 liquid for balancing for 3min, transferring the embryo into TS3 liquid for balancing for 5min, finally balancing for 5min in WS liquid, transferring the embryo into a culture medium, putting the culture medium into a culture box, evaluating the survival condition of the embryo after culturing for 2 hours, and then continuously culturing the surviving embryo until the blastocyst stage is incubated; the TS1 liquid, TS2 liquid, TS3 liquid and WS liquid are the same as in example 1.
Example 3
A stress-resistant cryopreservation method for a mouse embryo thinned by combining a zona pellucida comprises the following steps:
(1) preparation of anti-stress refrigerating fluid: the anti-stress refrigerating fluid comprises a freezing equilibrium fluid and a refrigerating fluid;
the frozen equilibrium liquid adopts HTF as a base liquid, 7% by volume of EG, 8% by volume of DMSO, 13% by volume of HSA, 250um/L vitamin C and 90um/L vitamin E are added;
HTF is adopted as a base liquid of the refrigerating fluid, 12% by volume of EG, 16% by volume of DMSO, 0.5M sucrose, 10% by volume of HSA, 250um/L vitamin C and 90um/L vitamin E are added;
(2) thinning of embryo zona pellucida: the same as example 1;
(3) putting the thinned embryo into a freezing balance liquid for balancing, transferring the embryo into a freezing liquid to clean and transfer among different liquid drops when the embryo is restored to 75 percent of the original volume, and putting the embryo on a carrying rod within 1 minute and quickly immersing the embryo into liquid nitrogen; the procedure was as in example 1.
The frozen mouse embryos were thawed as described above in example 1.
In order to verify the effect of the invention, a comparative example is set for comparison, which specifically comprises the following steps:
comparative example 1: using traditional programmed freezing, namely slow freezing, without thinning the transparent belt, and adding no vitamin E and vitamin C into the freezing liquid, and thawing according to a conventional method;
comparative example 2: using traditional vitrification freezing without thinning a transparent belt, adding no vitamin E or vitamin C into a freezing liquid, and unfreezing according to a conventional method;
comparative example 3: the same as example 1 except that: no thinning of the transparent tape is performed;
comparative example 4: the same as example 1 except that: vitamin E and vitamin C are not added in the anti-stress refrigerating fluid.
The statistics and comparison of the freezing equilibrium time, blastocyst formation rate, blastocyst hatching rate and thawing recovery rate were performed for the above four freezing methods and the method of example 1, and are specifically shown in table 1.
TABLE 1
The indices in table 1 illustrate:
freezing equilibrium time: the longer the time of in vitro balance, i.e. the longer the time of in vitro storage of the embryo, the greater the influence of potential harmful substances of the freezing fluid and the environment on the in vitro development potential of the embryo.
Thawing recovery rate: is a key index for measuring the freezing effect, and the low recovery rate indicates that the embryo is damaged and degenerated more during freezing, so that the embryo cannot be utilized, namely the utilization rate is low.
Blastocyst formation rate: is an index for measuring the continuous development ability of the thawed embryo, and the freezing method and the composition of the freezing liquid can have potential influence on the formation of the blastocyst.
Blastocyst hatching rate: the method is also an index for measuring the continuous development capability of the thawed embryo, is a result of the further development of the blastula, and can further reflect the influence of a freezing method, freezing liquid and an operating environment on the final development potential of the embryo compared with the blastula formation rate.
The differences of the anti-stress freezing method of the thinned zona pellucida in example 1 and the programmed freezing method in comparative example 1 in thawing recovery rate, blastocyst formation rate and blastocyst hatchability are tested by Chi's test, and the anti-stress freezing method of the thinned zona pellucida in example 1 and the programmed freezing method in comparative example 1 in thawing recovery rate (chi%257.8, P < 0.01) and high rate of blastocyst formation (χ)259.96, P < 0.01) and the hatching rate of the blastocyst is very obvious29.4, P < 0.01) are very significant. Differences were very significant in freeze equilibration time using one-way anova (F. 27741, P < 0.01). The thinning zona pellucida anti-stress freezing method is obviously superior to a programmed freezing method in freezing balance time, thawing recovery rate, blastocyst formation rate and blastocyst hatching rate.
The chi-square test is adopted for the differences of thawing recovery rate, blastocyst formation rate and blastocyst hatchability between the thinning zona anti-stress freezing method of example 1 and the vitrification freezing method of comparative example 2, the differences of thawing recovery rate (chi 2 ═ 4.3, P < 0.05) between the thinning zona anti-stress freezing method and the vitrification freezing method are significant, the differences of blastocyst formation rate (chi 2 ═ 6.61, P < 0.05) between the thinning zona anti-stress freezing method and the vitrification freezing method are significant, and the differences of blastocyst hatchability (chi 2 ═ 5.1, P < 0.05) between the thinning zona anti-stress freezing method and the vitrification freezing method are significant. Differences were very significant in freeze equilibration time using one-way anova (F153.9, P < 0.01). The thinning zona pellucida anti-stress freezing method is superior to a vitrification freezing method in freezing balance time, thawing recovery rate and blastocyst hatching rate.
The differences of thawing recovery rate, blastocyst formation rate and blastocyst hatching rate of the thinning zona pellucida anti-stress freezing method of example 1 and comparative example 3 are tested by chi-square method, the differences of the thinning zona pellucida anti-stress freezing method of comparative example 3 on thawing recovery rate (chi 2 is 4.02, P is less than 0.05) are obvious, the differences of the blastocyst formation rate (chi 2 is 0.15, P is more than 0.05) are not obvious, and the differences of the blastocyst hatching rate (chi 2 is 5.30, P is less than 0.05) are obvious. Differences were very significant in freeze equilibration time using one-way anova (F. 149.36, P < 0.01). The thinning zona pellucida anti-stress freezing method of example 1 is superior to comparative example 3 in freezing balance time, thawing recovery rate and blastocyst hatching rate, and shows that the method for thinning zona pellucida can greatly influence the freezing and thawing effects of mouse embryos and the development potential of the embryos.
The differences of thawing recovery rate, blastocyst formation rate and blastocyst hatching rate of the thinning zona pellucida anti-stress freezing method of example 1 and comparative example 4 are tested by chi-square method, the differences of the thinning zona pellucida anti-stress freezing method of comparative example 4 on the thawing recovery rate (chi 2 ═ 1.46, P > 0.05) are not significant, the differences of the blastocyst formation rate (chi 2 ═ 4.08, P < 0.05) are significant, and the differences of the blastocyst hatching rate (chi 2 ═ 6.50, P < 0.05) are significant. Differences were not significant using one-way anova (F ═ 2.7, P > 0.05) at the time of freeze equilibration. The anti-stress freezing method of the thinned zona pellucida in example 1 is superior to that in comparative example 4 in blastocyst formation rate and blastocyst hatching rate, and shows that the freezing and thawing effects of mouse embryos and the development potential of the embryos can be greatly influenced by adding a certain amount of vitamin E and vitamin C into the freezing solution and the thawing solution.
Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. A stress-resistant cryopreservation method for a mouse embryo thinned by combining a zona pellucida is characterized by comprising the following steps of:
(1) preparation of anti-stress refrigerating fluid: the anti-stress refrigerating fluid comprises a freezing equilibrium fluid and a refrigerating fluid;
the frozen equilibrium liquid adopts HTF as a basic liquid, and 7-8% by volume of EG, 7-8% by volume of DMSO, 11-13% by volume of HSA, 150-250um/L vitamin C and 90-100um/L vitamin E are added;
HTF is adopted as a base liquid of the refrigerating fluid, 12-16% by volume of EG, 13-16% by volume of DMSO, 0.5M sucrose, 10-15% by volume of HSA, 150-250um/L vitamin C and 90-100um/L vitamin E are added;
(2) thinning of embryo zona pellucida: thinning the zona pellucida of the embryo at the cleavage stage of the third day after fertilization in a mouse, taking the embryo at the 6-10 cell stage of the third day, marking the embryo by using a ruler, dividing the embryo into four regions on average, thinning the zona pellucida of one region in the middle of the zona pellucida by using a laser film-breaking instrument, wherein the total thinned region accounts for one fourth of the embryo, the pulse width of the laser is 0.200ms, the aperture is 2-3um, thinning is carried out by using continuous laser, the zona pellucida is thinned for one fourth in three times, the pulse track is adjusted to be matched with the radian of the zona pellucida during each thinning, 6 pulse laser thinning is carried out continuously each time, and finally, the zona pellucida is cut at two ends of the thinned region;
(3) putting the thinned embryo into a freezing balance liquid for balancing, transferring the embryo into a freezing liquid to clean and transfer among different liquid drops when the embryo is restored to 75-85% of the original volume, putting the embryo on a carrying rod within 1 minute, and quickly immersing the embryo into liquid nitrogen.
2. The method of claim 1, wherein the combination of zona pellucida thinning mouse embryo anti-stress cryopreservation method comprises: in the step (1), HTF is used as a base solution of the frozen equilibrium liquid, 7.5% by volume of EG, 7.5% by volume of DMSO, 12% by volume of HSA, 200um/L of vitamin C and 100um/L of vitamin E are added;
HTF is adopted as a base liquid of the refrigerating fluid, 15% by volume of EG, 15% by volume of DMSO, 0.5M sucrose, 12% by volume of HSA, 200um/L vitamin C and 100um/L vitamin E are added.
3. The method for cryopreserving a mouse embryo thinned by combining the zona pellucida as claimed in claim 1, wherein the specific operation steps of the step (3) are as follows:
s1, placing the embryo on the surface of the first frozen equilibrium liquid drop, allowing the embryo to freely fall to the bottom of the first frozen equilibrium liquid drop, and staying in the first frozen equilibrium liquid drop for 2 min; then transferring the embryo to the bottom of the second frozen equilibrium liquid drop and purging for 3 times, and staying in the second frozen equilibrium liquid drop for 1 min; then transferring the embryo to the bottom of the third frozen equilibrium liquid drop and blowing for 3 times, and observing the condition of the blastomere of the embryo;
and S2, when the embryo is recovered to 80% of the original volume, transferring the embryo into the freezing liquid drop, sucking the embryo to the bottom of the first freezing liquid drop, allowing the embryo to float to the upper part of the liquid surface, then quickly transferring the embryo to the bottom of the second freezing liquid drop for purging for 3 times, then quickly transferring the embryo to the carrying rod after again transferring to the bottom of the third freezing liquid drop for purging for 3 times, and placing the embryo into liquid nitrogen for freezing and storing.
4. The method of claim 3, wherein the combination of zona pellucida thinning mouse embryo anti-stress cryopreservation method comprises: in step S2, the retention time of the embryo in the freezing liquid is controlled within 60S.
5. A thawing method of mouse embryo is characterized in that: taking the frozen mouse embryo of any one of claims 1-4, rapidly inserting the carrying rod with the embryo into TS1 solution preheated to 37 ℃ for balancing for 1min, transferring the embryo into TS2 solution for balancing for 3min, transferring the embryo into TS3 solution for balancing for 5min, finally balancing in WS solution for 5min, transferring the embryo into a culture medium, placing the culture medium into an incubator, evaluating the survival condition of the embryo after culturing for 2 hours, and then continuing culturing the surviving embryo until the blastocyst stage till the blastocyst is hatched;
the TS1 liquid, the TS2 liquid, the TS3 liquid and the WS liquid are all prepared by taking HTF as base liquid, wherein 1M sucrose, 200um/L vitamin C and 12% HSA in volume percentage are added into the TS1 liquid; 0.5M sucrose, 200um/L vitamin C and 12% HSA by volume are added into the TS2 liquid; 0.25M sucrose, 200um/L vitamin C and 12% HSA by volume are added into the TS3 liquid; WS solution was supplemented with 200um/L vitamin C and 12% by volume HSA.
6. The method for thawing a mouse embryo according to claim 5, wherein the specific thawing procedures of the mouse embryo are as follows:
A. inserting the tail end of the carrying rod with the embryo into TS1 liquid preheated to 37 ℃, separating the embryo from the carrying rod by using a suction pipe, and staying in TS1 liquid for 1 min;
B. adding the embryo with TS1 liquid to the surface of the first TS2 liquid drop to allow the embryo to freely fall down and stay for 1min, then transferring the embryo to the bottom of the second TS2 liquid drop and blowing and sucking for 3 times and staying for 1min, and then transferring the embryo to the bottom of the third TS2 liquid drop and blowing and sucking for 3 times and staying for 1 min;
C. transferring the embryo with the TS2 liquid to the surface of a first TS3 liquid drop, allowing the embryo to fall freely and stay in the liquid drop for 1min, then transferring the embryo to the bottom of a second TS3 liquid drop and sucking for 3 times and staying for 2min, and then transferring the embryo to the bottom of a third TS3 liquid drop and sucking for 3 times and staying for 2 min;
D. transferring the embryo to the bottom of the first WS droplet for 3 times, transferring the embryo to the second WS droplet for 2min, transferring to the bottom of the third WS droplet for 3 times, staying for 3min, and transferring to a blastocyst culture medium for continuous culture.
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