CN217429092U - Oocyte and embryo vitrification freezing carrier - Google Patents
Oocyte and embryo vitrification freezing carrier Download PDFInfo
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- CN217429092U CN217429092U CN202123165898.2U CN202123165898U CN217429092U CN 217429092 U CN217429092 U CN 217429092U CN 202123165898 U CN202123165898 U CN 202123165898U CN 217429092 U CN217429092 U CN 217429092U
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Abstract
The utility model provides an oocyte and embryo vitrification freezing carrier, which belongs to the technical field of animal embryo engineering. Oocyte and animal embryo vitrification freezing carrier includes the cryovial body, the one end of cryovial body is U type bevel connection the inner wall of the U type bevel connection end of cryovial body is pasted to have water absorbing material. After the refrigerating fluid containing the oocytes and/or embryos is transferred to the water absorbing material, redundant refrigerating fluid can be automatically absorbed, the volume of the final refrigerating fluid is controlled to be about 0.5 mu l, and the refrigerating efficiency is greatly improved. Adopt the utility model discloses a freezing carrier to carry out vitrification freezing to animal oocyte and/or embryo, the recovery rate of embryo can reach more than 99%.
Description
Technical Field
The utility model belongs to the technical field of animal embryo engineering, concretely relates to oocyte and embryo vitrification freezing carrier.
Background
The vitrification freezing of oocyte and embryo is to dehydrate oocyte and embryo serially and to set the dehydrated oocyte and embryo on one carrier and to feed the carrier into liquid nitrogen environment at-196 deg.c for fast cooling to make the liquid inside and outside the cell reach vitrification state. Oocyte and embryo freezing is one of the important methods for preserving animal genetic resources. Vitrification freezing is to treat oocyte and embryo with relatively high concentration and very viscous freezing protectant to freeze the embryo from physiological temperature to-196 deg.c and to freeze the liquid inside and outside the embryo cell into vitrified solid state. Compared with a program freezing method, in the freezing process of the vitrification method, liquid in the embryo cells can not form ice crystals, so that physical and chemical damages of the ice crystals to the oocyte and the embryo cells are avoided, and a better freezing effect is obtained. The recovery rate of the vitrified frozen embryo is usually more than 95%, if the blastocyst is frozen, the effect is better, the recovery rate can be more than 99%, the recovery rate is obviously higher than that of a program freezing method, and the advantages are obvious, so that more and more centers gradually start to use the vitrified freezing method and become the main method for embryo freezing at the present stage. Likewise, vitrification freezing has become the gold standard for cryopreservation of oocytes.
The center of the prior art discloses a variety of embryo vitrification freezing vehicles, such as open elongated tubules (OPS) and cryotops. However, the survival rate and conception rate of the embryos vitrified frozen by OPS and Cryotop after thawing are not good. This is because the volume of the final freezing fluid affects the cooling and heating speed of the embryo freezing/thawing process, thereby determining the final freezing effect. For example, the final freezing liquid volume of OPS is 2-5 μ l, and the freezing volume is large. The final refrigerating fluid volume of Cryotop can be controlled to be about 1 mul, but the excessive fluid needs to be sucked manually, which needs years of training and working experience of operators to complete the complicated step, so the operation effect is different and unstable.
SUMMERY OF THE UTILITY MODEL
In view of this, the utility model aims at providing an oocyte and embryo vitrification freezing carrier can control final refrigerating fluid volume at 0.3 ~ 0.8 mul, has improved freezing efficiency greatly.
The utility model provides an oocyte and embryo vitrification carrier that freezes, including the cryovial body, the one end of cryovial body is U type bevel connection, its characterized in that the inner wall of the U type bevel connection end of cryovial body is pasted with water absorbing material.
Preferably, the water absorbent material comprises filter paper.
Preferably, the shape of the water absorbing material comprises a rectangle; the length of the water absorbing material is 18-20 mm, and the width of the water absorbing material is 5-6 mm.
Preferably, the outer edge of the water absorbing material is flush with the outer edge of the U-shaped bevel opening.
Preferably, the angle of the oblique angle of the U-shaped oblique opening is 60 degrees.
Preferably, the length of the U-shaped bevel opening is 0.5-1 cm
Preferably, the freezing pipe body is prepared by beveling one end of a semen freezing pipe; the specification of the semen freezing tube is 0.25 mL.
Preferably, the water absorbing material is inserted into the freezing pipe body from the U-shaped inclined opening, and naturally attached to the inner wall of the freezing pipe body without being adhered by an adhesive.
The utility model provides an oocyte and embryo vitrification carrier that freezes, including the cryovial body, the one end of cryovial body is U type bevel connection the inner wall facing of the U type bevel connection end of cryovial body has water absorbing material. After the refrigerating fluid containing the oocyte and/or embryo is transferred to the water absorbing material, redundant refrigerating fluid can be automatically absorbed, so that the volume of the final refrigerating fluid can be controlled to be about 0.5 mu l, the refrigerating efficiency and the recovery rate of the frozen embryo are greatly improved, and the carrier is named as 'Paper Straw' or 'Paper tube'. Adopt the utility model discloses a freezing carrier to carry out vitrification freezing to animal oocyte and/or embryo, the recovery rate of embryo is more than 99%. In addition, adopt the utility model discloses a carrier carries out vitrification freezing to animal oocyte and/or embryo, easy operation, and the technical staff need not train alright use, for vitrification freezing technology commercialization provides the technical guarantee, has enlarged the wide application of this technique in the livestock breeding greatly.
Drawings
FIG. 1 is a schematic diagram of the structure of a semen freezing tube, wherein 1-semen freezing tube body, 11-cotton plug;
FIG. 2 is a schematic diagram of a bevel cutting of a semen freezing tube, wherein 1-the semen freezing tube body, 11-a cotton plug, 3-a scalpel, 4-U-shaped bevel length and 5-U-shaped bevel;
FIG. 3 is a schematic view of a water absorbing material inserted into a freezing tube body from a U-shaped bevel, wherein 1 is the semen freezing tube body, 11 is a cotton plug, 2 is the water absorbing material, and 5 is the U-shaped bevel;
FIG. 4 is a schematic diagram of the freezing liquid containing the oocyte and/or embryo added on the water-absorbing material of the oocyte and animal embryo vitrification freezing carrier, wherein, 1-semen freezing tube body, 11-cotton plug, 2-water-absorbing material, 5-U-shaped bevel opening and 6-freezing liquid containing the oocyte and/or embryo.
Detailed Description
The utility model provides an oocyte and embryo vitrification freezing carrier, including the cryovial body, the one end of cryovial body is U type bevel connection, its characterized in that the inner wall of the U type bevel connection end of cryovial body is attached with water-absorbing material.
The utility model discloses an oocyte and embryo vitrification freezing carrier is applicable to people and animal.
In the utility model discloses in, what water-absorbing material was preferred includes filter paper, and filter paper is economic cheap, the utility model discloses an oocyte and embryo vitrification freezing carrier can reach the refrigeration effect comparable with Cryotop, but more economical and practical, the cost is lower, and Cryotop's selling price is $ 20, and the utility model discloses an embryo vitrification freezing carrier cost closes $ 0.5 about. In the present invention, the shape of the water absorbing material preferably includes a rectangle; the length of the water absorbing material is preferably 18-20 mm, and the width of the water absorbing material is preferably 5-6 mm. The utility model discloses in, the outer edge of water absorption material and the outer edge parallel and level of U type bevel connection.
In the utility model, the angle of the oblique angle of the U-shaped oblique opening is preferably 60 degrees; the length of the U-shaped bevel opening is 0.5-1 cm.
In the present invention, the method for preparing the oocyte and embryo vitrification freezing carrier preferably includes the following steps: beveling one end of the semen freezing pipe to form a freezing pipe body with a U-shaped bevel opening; and a water absorbing material is attached to the inner wall of the U-shaped beveled end of the freezing pipe body.
In the present invention, the beveling is preferably performed using a scalpel; the specification of the bevel cutting semen freezing tube is preferably 0.25mL or 0.5mL, the structural schematic diagram of the semen freezing tube is shown in figure 1, and the bevel cutting schematic diagram is shown in figure 2.
The utility model discloses in, the material that absorbs water inserts the freezer pipe from U type bevel connection originally internally, and natural adhesion is on the inner wall of freezer pipe body, and the adhesion of adhesion agent is not passed through. In the present invention, the schematic diagram of the water absorbing material inserted into the freezing pipe body from the U-shaped bevel is shown in fig. 3.
The using method of the oocyte and embryo vitrification freezing carrier of the utility model preferably comprises the following steps:
transferring the freezing liquid containing the oocytes and/or embryos to the water absorbing material of the vitrification freezing carrier in the scheme, and directly throwing the front end of the freezing liquid into liquid nitrogen for freezing. The frozen vector was then stored in a common liquid nitrogen tank. See figure 4 for a schematic illustration.
The oocyte and embryo vitrification freezing carrier of the utility model is preferably sterilized before use; the sterilization mode is preferably gas sterilization, and more preferably ozone sterilization.
In the present invention, the time for transferring is preferably 5 to 10 seconds before the oocyte and the embryo are frozen by the freezing liquid.
The utility model has no special limitation to the formula of the refrigerating fluid, and can be prepared by adopting the conventional vitrification refrigerating fluid and the refrigeration method in the field.
After the oocyte and the embryo are vitrified and frozen, the utility model preferably also comprises unfreezing; the thawing method comprises the following steps: adding the vitrified frozen carrier into the thawing solution for thawing. The utility model has no special limitation to the formula of the thawing solution and the thawing parameters, and can be applied to the common vitrification thawing solution in the field.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1
1) Cutting off the upper part of a thin tube at an angle of 60 degrees at a position of 1.0cm at the front end of a semen freezing tube with the specification of 0.25ml by using a scalpel, so that a U-shaped groove inclined opening is formed at the bottom of the thin tube;
2) inserting chemical filter paper with the length of 20mm and the width of 5mm into a thin tube with an inclined opening of the U-shaped groove, and enabling the front end of the filter paper to be flush with the front end of the thin tube; filling 10-15 paper tubes into a gas sterilization bag, and performing gas sterilization to obtain a sterilized vitrified freezing carrier (paper tube);
3) and 5-10 s before the oocyte and the embryo are subjected to freezing treatment by the refrigerating fluid, transferring the refrigerating fluid containing the oocyte and/or the embryo to the water absorption material of the paper tube obtained in the step 2), and transferring the water absorption material into liquid nitrogen for freezing.
And directly putting the paper tube into the thawing solution for thawing.
Example 2
1. Retrieval of oocytes
The method comprises the steps of carrying out intraperitoneal injection on a 10 IU/PMSG (Sigma) mouse (a Swiss mouse and a B6D2 hybridized F1 mother mouse) on a 8-10-week-old mother mouse, and carrying out intraperitoneal injection on 100 IU/HCG after 48-50 h. And (3) carrying out cervical dislocation and killing 18-20 h after HCG (Sigma) injection, taking out the oviduct, placing the oviduct in a culture solution M2(Sigma), dissecting under a microscope, scratching the expanded part of the oviduct by using a syringe needle to see a cumulus complex, transferring the cumulus complex into a hyaluronidase solution (Sigma) containing 80 IU/ml for digestion, repeatedly blowing, sucking and degranulating, collecting oocytes with normal shapes and first polar bodies, placing the oocytes into an M16(Sigma) solution, covering, and placing the oocytes in a 5% incubator at 37 ℃ for later use.
2. Embryo acquisition
By using the mouse super-volley scheme, 8-10-week-old female mice (Swiss mice and B6D2 are hybridized to obtain F1 female mice) are subjected to super-volley treatment by intraperitoneal injection of PMSG and HCG, and then are caged with male mice in a ratio of 1: 1. And (3) closing the cages and viewing the suppository, then dislocating and killing the cervical vertebra of the donor mouse, soaking and disinfecting the donor mouse by using alcohol, shearing the abdominal skin in an anatomical disc and expanding the abdominal skin upwards to expose the abdominal wall muscles, shearing the muscles at the front edge of the pubis, poking the internal organs, and exposing the uterine horn. The fat on the uterine horn is stripped off by an ophthalmic scissors. After the cervix is cut, the uterine horn is removed along with the fallopian tube and ovary and placed in a 50mm petri dish. The uterine horn and the fallopian tube junction are opened by a No. 5 thin needle, 1-2 mL of DPBS (Sigma) + 5% BSA solution (37 ℃) is extracted by an injector, a special flat head injection needle is connected, and the uterine horns on two sides enter the uterine body respectively from the uterine body to perform embryo flushing. After the liquid is flushed out and kept stand for a while, the embryo in the well-developed blastocyst stage is picked up by an egg picking needle under a stereomicroscope, and then is put into M16 liquid and is placed in a 5 percent incubator at 37 ℃ for standby.
3. Vitrification freezing procedure
The frozen base Solution (HS) was calf blood (Fetal CalfSerum, FCS) 10% (v/v) + DPBS (Sigma). Firstly, pre-balancing oocytes or embryos in a basic Solution for 2min at room temperature, then transferring the oocytes or embryos into a balancing Solution (ES), and balancing the basic Solution containing 7.5% (V/V) of dimethyl sulfoxide (DMSO) and 7.5% (V/V) of Ethylene Glycol (EG) for 3 min; finally, in a vitrified refrigerating fluid (VS): equilibrium 90s (oocytes), or 45 (embryos) in a base solution of DMSO 15% (V/V) + EG 15% (V/V) +1.0M sucrose. According to experimental requirements, oocytes or embryos are loaded on different vitrification freezing carriers (OPS, Cryotop and paper tubes) and directly put into liquid nitrogen for freezing.
4. Thawing procedure
Before the recovery of the oocyte or embryo, the thawing solution is heated to room temperature, the freezing carrier is taken out from liquid nitrogen, the front end of the freezing carrier is placed in the thawing solution 1 (base solution +1.0M sucrose) after being exposed in the air for 3s, so that the oocyte or embryo is fallen into the liquid, and after the balancing for 1min, the freezing carrier is sequentially transferred to the thawing solution 2 (base solution +0.5M sucrose), the thawing solution 3 (base solution +0.25M sucrose), and the freezing carrier is respectively 3min in the base solution. Performing in vitro fertilization on the unfrozen oocyte, and observing the fertilization rate and the blastocyst development rate; the thawed embryos will continue to be cultured for survival, development and hatchability.
5. Oocyte In Vitro Fertilization (IVF) and development
The thawed oocytes were transferred to recipient fluid (HTF, Sigma) at 37 ℃ in 5% CO 2 Fertilization was carried out after 2h of subculture. During the period, the male mice of 10-14 weeks are killed by cervical dislocation, the abdomen is cut open to expose the tissues of the male mice testis and epididymis, the epididymis is separated, the semen is taken out after puncture/shear, the obtained mixture is placed in a TYH (Sigma) culture drop and placed at 37 ℃ and 5% CO 2 And (5) incubating in an incubator to enable the sperms to acquire energy for 1-2 hours. Observing sperm motility and state under optical microscope, dropping appropriate amount of sperm into the incubated oocyte culture drop according to sperm state, and performing in vitro fertilization (fertilization density of 2 × 10) 5 Sperm/ml). After about 4h after fertilization, the culture solution is replaced, embryos are washed 3 times with M2, transferred into M6 culture solution and continuously placed in CO 2 Culturing in an incubator. And (4) counting the recovery rate and the cleavage rate 16-24 hours after fertilization, and counting the blastocyst rate 5 days after fertilization. Hatchability was counted on day 6 after fertilization.
The recovery rate is survival number of the viable oocyte/number of the unfrozen oocyte multiplied by 100 percent;
the cleavage rate is equivalent to fertilized egg number/total number of eggs × 100%.
Blastocyst rate is blastocyst number/total oocytes × 100%.
Hatching rate is the number of hatching blastocysts/total blastocysts multiplied by 100%.
6. Development of embryo
Putting the thawed blastocysts into M16 solution, and placing the blastocysts in a 5% incubator at 37 ℃ for further culture for 48h for observation. Statistical resuscitation and blastocyst rates were recorded at 24 h. Statistical blastocyst hatchability was recorded at 48 h.
The recovery rate is the survival rate of blastula/the number of thawed blastula multiplied by 100 percent;
the blastula rate is the blastula number/total thawed embryo number multiplied by 100 percent;
the blastocyst hatching rate is the number of hatched blastocysts/total blastocysts multiplied by 100%.
Experimental design and results
1. Biometric method
Statistical methods statistical data were performed using SPSS11.5 software, and the counts were tested by χ 2, with the threshold for significance differences set at P < 0.05. Different letters represent significant differences.
2. Vitrification freezing of oocyte
TABLE 1 Effect of different vitrification vectors on oocyte freezing, in vitro fertilization and embryo development
3. Embryo vitrification freezing
TABLE 2 Effect of different vitrification vectors on freezing and its embryonic development
Although the above embodiments have been described in detail, it is only a part of the embodiments of the present invention, and not all embodiments, and people can obtain other embodiments without inventive step according to the present embodiments, and these embodiments all belong to the protection scope of the present invention.
Claims (8)
1. The utility model provides an oocyte and embryo vitrification carrier that freezes, includes the cryovial body, the one end of cryovial body is the oblique mouth of U type, its characterized in that the inner wall of the oblique mouth end of U type of cryovial body is pasted and is had water absorption material.
2. The oocyte and embryo vitrification freezing vehicle of claim 1 wherein the water absorbing material includes filter paper.
3. An oocyte and embryo vitrification freezing carrier according to claim 1 or 2, characterized in that the shape of the water absorbing material includes a rectangle; the length of the water absorbing material is 18-20 mm, and the width of the water absorbing material is 5-6 mm.
4. The oocyte and embryo vitrification freezing carrier according to claim 3, wherein the outer edge of the water absorbing material is flush with the outer edge of the U-shaped bevel.
5. The oocyte and embryo vitrification freezing carrier according to claim 1, wherein an angle of an oblique angle of the U-shaped oblique incision is 60 °.
6. The oocyte and embryo vitrification freezing carrier according to claim 1 or 5, wherein the length of the U-shaped bevel is 0.5-1 cm.
7. The oocyte and embryo vitrification freezing carrier according to claim 1, characterized in that the freezing tube body is prepared by beveling one end of a semen freezing tube; the specification of the semen freezing tube is 0.25 mL.
8. The oocyte and embryo vitrification freezing carrier according to claim 1, characterized in that the water absorbing material is inserted into the freezing pipe body from a U-shaped bevel opening, naturally attached to the inner wall of the freezing pipe body, and not adhered by an adhesive.
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| CN114027295A (en) * | 2021-12-16 | 2022-02-11 | 草原和牛投资有限公司 | Oocyte and embryo vitrification freezing carrier and method |
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| CN114027295A (en) * | 2021-12-16 | 2022-02-11 | 草原和牛投资有限公司 | Oocyte and embryo vitrification freezing carrier and method |
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