RU2707252C1 - Method for cattle oocyte freezing - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology. This method for vitrification of oocytes of mammals can be successfully applied to agricultural animals, in particular for cattle. Oocytes from cattle are recovered by aspiration from antral follicles, conducting morphological selection and washing with physiologic saline. It is then frozen in series in three vitrification solutions containing a combination of cryoprotectors and excipients, after which content with oocytes is placed in a cryotube partially submerged into liquid nitrogen, and after tightening the cap, the cryotube is immersed into a container with liquid nitrogen and then into a Dewar vessel. Disclosed method of freezing oocytes of cattle is less toxic due to reduction of cryoprotectors, invention allows protect a considerable part of oocytes from damages.EFFECT: as a result of this vitrification procedure efficiency of revitalization of oocytes after thawing in more than 80 % of cases is achieved.7 cl, 9 dwg, 2 tbl

Description

The invention relates to biotechnology in agriculture and animal husbandry, in particular, to the field of cryopreservation of biological objects, and can be used to freeze cattle oocytes by vitrification.

Over the course of three decades, the popularity of embryo transplantation has been growing steadily. She received a “second wind” with the discovery of opportunities: replacing infectious trade in pedigree cattle with biotechnological export-import of pedigree resources, obtaining sexually-sexed (ordered sex) embryos, the technology of washing eggs (oocytes), their in vitro fertilization and growing outside the body ( in vitro). The technology of cryopreservation of oocytes and embryos, as well as the creation of national banks of hereditary material of breeding animals will significantly reduce the cost of purchasing imported cattle, increase livestock by increasing the reproduction rate, which will improve the profitability of breeding farms. Export of purebred breeding cattle from Russia is practically not carried out. This problem can also be solved by exporting frozen oocytes or cattle embryos.

The problem of preserving the hereditary material of valuable breeds of domestic animals has not yet been solved. Various methods of cryopreservation (vitrification) of biological material (gametes and tissues) are offered on the market, but in most cases, after thawing, a significant decrease in cell viability and functional activity is recorded. The question of the effectiveness of cryoprotectants and their effect on the safety of DNA, cell membranes, organelles during freezing and long-term storage at ultra low temperatures remains relevant.

The standard method for freezing oocytes for humans and animals is slow freezing using a medium supplemented with animal components (cattle serum, albumin) and various cryoprotectants: propanediol (PD), dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG) in various combinations and concentrations. Cryoprotectants PD, DMSO, EG and PG are able to penetrate the membranes of oocytes and stromal cells, they protect the cellular structures both inside and around the cells. These penetrating cryoprotectants are often used in combination with non-penetrating ones such as sucrose, glucose, glycerin or serum albumin. Slow freezing leads to the formation of ice crystals and damage to the structure of the oocyte and reduces its viability. It is believed that if the freezing rate is significantly increased, and, consequently, the contact time of the biomaterial with cryoprotectants is reduced to avoid a toxic effect, then it is possible to achieve greater survival of thawed (revitalized) cells and tissues.

There are only a few foreign companies that present their products for freezing by vitrification on the Russian market. The first in the development of a method for vitrification of oocytes were Japanese scientists. Based on the highly effective method of vitrification of eggs, the first set of Cryotop reagents was created (Kitazato, Japan), but it contains proteins of animal origin and antibiotics. The Japanese company CryoTech Lab also produces a similar kit for vitrification of oocytes and embryos. However, it contains only one cryoprotectant and polysaccharide, as well as an antibiotic and albumin. Belgian company FertiPro N.V. Launches VitriFreeze / ThawES Kit, it contains human albumin, although it does not contain antibiotics. The Medicult Vitrification Cooling Reagent Kit (Origio, USA) does not contain DMSO, but includes human albumin. All of these kits are designed for freezing by vitrification of human oocytes and embryos, and not for vitrification of egg and animal embryos.

In the Russian database of inventions, there are several methods, among which are installations for vitrification of mammalian oocytes and embryos, environments for culturing mammalian animal and human follicles, methods for cryopreservation of marine invertebrate cells, sex cells of cattle and sperm of cattle, described cryoprotectants for vitrification of mammalian oocytes and embryos (mice, pigs). Closest to the proposed technical solution are examples and the invention described in the following three patents.

The known "Method of culturing in vitro ovarian follicles" according to the patent RU 2286384. The invention is based on a method for producing a large number of mature or immature follicles and / or oocytes from mammary ovary tissue (mice). In their invention, scientists describe an example of the creation and use of a follicle bank. This method describes that mouse follicles are collected in plastic cryo-ampoules (Simport, Quebec, Canada), 25 follicles per ampoule are suspended in 150 μl of L15 medium with 10% heat inactivated fetal calf serum and 1.5 M DMSO. Then slowly freeze using a programmable freezer (Cell Freezer R204; Planer, Sunbury-on-Thames, UK). The follicles are kept in a freezing mixture for 15 minutes at 4 ° C, then cooled to -7 ° C at a rate of 2 ° C per minute. After manual transfer, the temperature is reduced to -40 ° C at a rate of -0.3 ° C per minute before being placed in liquid nitrogen, where the follicles very quickly cooled to -110 ° C at a rate of -50 ° C per minute. Follicles are thawed very quickly by heating cryo-ampoules to 37 ° C. The cryoprotectant is dissolved in three stages (reduce the concentration of DMSO from 1.5 M to 1 M, then to 0.5 M) for 15 minutes at room temperature. Before cultivation, the follicles are incubated for 15 min at 37 ° C in a medium for isolation.

The disadvantages of this method is the slow freezing, and not vitrification. Follicles from the tissue of the ovaries of mammals (mice), rather than oocytes, are used as an object. The authors use calf serum, which increases the risk of possible infection of biological material. The method of slow freezing requires the use of additional equipment (programmable freezing apparatus) and does not protect cells from the damaging effects of ice crystals. Also, the method is suitable only for slow freezing of mouse follicles, and not CRC oocytes, which differ in a number of physiological parameters.

The known method "System and device (options) for cryopreservation of a group of oocytes and mammalian embryos according to the method of vitrification when used as a carrier of hollow fiber" according to patent RU 2584584. This invention may find application for cryopreservation of mouse and pig oocytes. The method is based on the description of a device for loading, vitrifying and heating a group of mammalian embryos when used as a carrier of hollow fiber. The device consists of a segment of a hollow fiber and a capillary with a cone-shaped tip, characterized in that the segment of the hollow fiber is attached directly to the disposable capillary by putting a segment of the hollow fiber on the conical tip. In an example of use, the inventors describe a method of vitrification by ultrafast cooling of an object pretreated in solutions with a high concentration of cryoprotectants, such as, for example, dimethyl sulfoxide (DMSO), ethylene glycol (EG), and sucrose. Technically, an ultrahigh cooling rate was achieved by direct immersion in a liquid nitrogen of a vitrified object, the total volume of which did not exceed 0.10 μl. The base solution for vitrification and heating of the embryos was Tyrode's medium with the addition of 20.0% of blood serum.

The disadvantages of this method is the use by inventors of cryoprotectants in high concentrations in combination with blood serum, which contains proteins of animal origin and can be a source of infection. Also, the method is suitable only for vitrification of mouse and pig oocytes, and not cattle oocytes, which differ in a number of parameters. The use of hollow fiber complicates the vitrification procedure and makes it longer in time, which can adversely affect the quality of oocytes and embryos after thawing.

Known "Method of low-temperature preservation of embryos" according to patent SU 1683621. This invention may find application for long-term storage of reproductive cells of mammals (mice). In it, a mixture of cryoprotectors: glycerol, propylene glycol, dimethyl sulfoxide and a 0.4% solution of choline chloride, is added to the embryos of mice obtained at the blastomere stage, on standard Dulbecco phosphate-salt medium to a final concentration of 4: 75-5.5% each. All procedures are carried out at a temperature of 4-8 ° C. After balancing, the embryos are poured into containers, rolled up and immersed for 5-10 seconds. into zinc powder, cooled to a temperature of liquid nitrogen (-196 ° C). Then the containers are transferred to liquid nitrogen.

This method does not apply to the cryopreservation of mammalian oocytes, but describes the procedure for preserving embryos. The main disadvantages are the implementation of all procedures at 4-8 ° C and the use of the method for vitrification of already fertilized mouse oocytes, i.e. embryos, not cattle oocytes.

The objective of the invention is to eliminate all these disadvantages, namely the use of a combination of cryoprotectants for cryopreservation of cattle oocytes in experimentally selected conditions. The present invention allows to get rid of the formation of ice crystals destroying the cell during vitrification (direct immersion of biological material in liquid nitrogen), since there is a substitution of water inside the cell for special substances - cryoprotectants, so the method is simpler to use and more effective. The proposed method does not require special devices for cryopreservation of biological objects, which makes the method simple and makes it less costly.

The technical result.

The present invention allows vitrification of cattle oocytes and obtain animal eggs of a high degree of viability, with confirmed biological activity, high stability after prolonged storage in liquid nitrogen and subsequent revitalization (thawing). This method is designed for long-term storage at ultra-low temperatures of sex cells (oocytes) of cattle, in order to ensure the safety of breeds, reduce the risk of losing the breeding core from infections, increase the speed of reproduction of the livestock.

The essence of the invention includes the extraction from the ovaries of cattle delivered to the laboratory for several hours at a temperature of 23-25 ° C, oocytes by aspiration from antral follicles more than 2 mm in diameter, then morphological selection of oocytes for subsequent freezing. The selected oocytes are subjected to rapid freezing by the method of vitrification, which is carried out under aseptic conditions (in a laminar box). Oocytes are placed in a solution based on HEPES buffer for 1-3 minutes, then they are sequentially transferred first to vitrification solution 1 containing a combination of cryoprotectants (EG and DMSO) at a concentration of 5-20% for 10-12 minutes. Next, the oocytes are moved for 1-2 min from solution 1 to solution 2, containing a combination of the same cryoprotectants in an increased concentration of 20-40%. Then the oocytes are moved for 20-40 s. from solution 2 to solution 3, additionally containing a non-penetrating cryoprotectant (sucrose) at a concentration of 1-10 M. Then from solution 3, the contents with oocytes in a volume of 5-50 μl are transferred to a cryovial partially immersed in liquid nitrogen, then the cap is tightly screwed and immersed cryovial into a container with liquid nitrogen. Next, a cryovial is transferred from a container with liquid nitrogen to a Dewar vessel or cryostorage with liquid nitrogen for long-term storage. As a result of this vitrification procedure, oocyte revitalization efficiency is achieved in more than 80% of cases. The proposed method of quick freezing can be further used to create national banks of hereditary material, namely cattle oocytes, as well as for subsequent thawing of oocytes, their in vitro fertilization, cultivation and subsequent transplantation of embryos.

List of drawings

FIG. 1 - Aspiration of antral follicles from the ovaries of cows.

FIG. 2 - Oocytes of cows in solution for isolation.

FIG. 3 - Tablet with vitrification media.

FIG. 4 - Diagram of filling the holes of a 4-well plate with a sterile syringe.

FIG. 5 - Immersion of a cryovial in a container with liquid nitrogen.

FIG. 6 - Transfer of a cryovial with oocytes after vitrification into a Dewar vessel.

FIG. 7 - General scheme of the procedure for freezing oocytes.

FIG. 8 - Class A cows oocytes prior to vitrification.

FIG. 9 - Cow oocytes (oocytes with normal morphology - A, degenerative oocytes - B) after vitrification and revitalization.

Table 1 - Classification of oocytes by morphology before freezing.

Table 2 - Results of oocyte survival after vitrification in media with different concentrations of cryoprotectants followed by revitalization.

The procedure for implementing the method

1. Biomaterial (ovaries of cows) should be delivered to the laboratory for several hours at room temperature. Oocytes can be extracted from the ovaries by aspiration with a syringe from antral follicles larger than 2 mm in diameter (Fig. 1). Wash the eggs with saline with HEPES buffer. Make a morphological selection of oocytes for subsequent freezing (Fig. 2).

2. Check the flushing medium and vitrification solutions for transparency, absence of suspensions or turbidity. Carefully plan the execution of all necessary manipulations. Media should always be used in laminar flow only to avoid contamination. Warm all media to room temperature (20-25 ° C) to the extent necessary for the freezing procedure. Prepare tweezers and a container with liquid nitrogen.

3. Using a sterile syringe, fill the wells of a 4-well plate (Fig. 3) in accordance with the following scheme (Fig. 4):

• add 300-500 μl of HEPES buffer to the first well of the tablet (any medium with HEPES or MOPS buffer),

• in the second well - 300-500 μl of Solution 1 containing a combination of cryoprotectants: EG and DMSO,

• in the third - 300-500 μl of Solution 2 containing an increased concentration of cryoprotectants: EG and DMSO,

• in the fourth - 300-500 μl of Solution 3, containing EG, DMSO and non-penetrating cryoprotectant - sucrose.

4. Next, prepare the required number of cryovials and sign, bearing in mind that up to 5 oocytes can be frozen in 1 cryovial. In one prepared portion of the medium, up to 5 vitrification cycles can be carried out for one individual animal.

• Under aseptic conditions (laminar box), place oocytes in the first well in the HEPES buffer for 1-3 minutes.

• Transfer oocytes from well 1 to well 2 for 8-12 minutes. After transferring the oocytes from the HEPES buffer (Hole No. 1) to Solution 1 (Hole No. 2), the oocytes will begin to change shape and shrink, and then return to their original size, this process takes no more than 12 minutes.

• Transfer oocytes from well 2 to well 3 for 1-2 minutes.

• Move oocytes from well 3 to well 4 for 20–40 s.

5. Holding the upper end of the open cryovial with tweezers, partially immerse it in liquid nitrogen, insert the tip of the dispenser tip into the cryovial and drip the contents with 1-5 oocytes. Then, holding the cryovial with tweezers with one hand, tighten the cap with the other hand and immerse the cryovial in a container with liquid nitrogen (Fig. 5). The complete procedure, including placing the oocyte on Wednesday Solution 3, loading the oocyte into a cryovial and sealing it, should take no more than 60 s. before immersion in liquid nitrogen. If this process takes more than 60 s, the result may deteriorate and oocyte survival will decrease.

6. Transfer all cryovials from a container with liquid nitrogen to a Dewar vessel or cryostorage with liquid nitrogen for long-term storage (Fig. 6).

Method implementation examples

Example 1. The selection of oocytes according to morphological criteria for vitrification.

As a result of aspiration of the antrum follicles of the ovaries of 20 cows of different age categories (from 2 to 9 years), 255 eggs of different maturity were obtained. On average, 12 oocytes were obtained from one cow. Of all the oocytes received, degenerative cells were 30.5%. For the vitrification procedure, only good morphology oocytes were selected, surrounded by cumulus cells (COCs), which corresponded to class A (Fig. 8).

After aspiration, COCs were washed in physiological saline or in phosphate-buffered saline (PBS) supplemented with a HEPES buffer and counted according to morphology, where class A corresponded to oocytes from all sides surrounded by cumulus cells, B partially surrounded, C uncoated oocytes, D - with altered cumulus cells. In accordance with this classification, all oocytes were distributed as follows as shown in table 1. Class A COCs were selected for the vitrification procedure.

Example 2. Survival of cattle oocytes in various environments.

In the course of our work, we compared the survival of COCs in cattle in environments with EG and in the combination of EG with DMSO. To assess oocyte survival, 32 COCs were previously thawed, pre-vitrified in an EG and DMSO solution (n = 20) and only in an EG solution (n = 20). Quick thawing of oocytes was performed by incubating successively in two FSB-based solutions containing sucrose in a concentration of 0.5-5 M for 3-5 minutes each with two dilutions. It was shown that the survival rate for vitrified oocytes in the EG was lower (70%) than in the combination of EG and DMSO (80%) (table 2). As a result, it was shown that for cattle oocytes it is more efficient to carry out the vitrification procedure in an environment with different cryoprotectants of EG and DMSO, using them in combination.

Example 3. Comparison of vitrification and slow freezing.

Cattle oocytes were frozen by vitrification and by slow freezing in a solution containing DMSO at a concentration of 5-15%. After storage in liquid nitrogen for 2-14 days, oocytes were thawed and revitalized. Quick thawing of oocytes was performed by incubating successively in two sucrose solutions at different concentrations of 3-5 min in each. After vitrification in cryopreservation media with different concentrations of EG with DMSO (Solution 1, 2, and 3), morphological evaluation of cow eggs showed that more than 75% of oocytes had normal morphology (Fig. 9 - A). And after slow freezing in a commercial solution with DMSO and subsequent thawing, there were structural changes in the morphology characteristic of degenerative oocytes in 60% of cases (Fig. 9 - B).

Confirmation of the achievement of a technical result.

The proposed method for freezing cattle oocytes is simple, since it does not require the use of additional equipment and is fast, since the method is based on the method of vitrification, rather than slow freezing. The method is the least toxic due to the use of a combination of cryoprotectants in reduced concentrations, the invention allows to protect a significant part of the oocytes from damage during freezing, which led to the achievement of the effectiveness of revitalization in more than 80% of cases. The method is characterized by the use of a serum-free medium containing no animal protein, which avoids the possible infection of biological material and makes the product suitable for various types of mammals. The absence of antibiotics in the proposed product has a positive effect on the quality of biological material.

Claims (7)

1. A method for freezing cattle oocytes, including extraction of oocytes from cows' ovaries by aspiration from antral follicles, morphological selection and quick freezing of oocytes by vitrification, characterized in that the oocytes are first placed in 300-500 μl with 1- HEPES buffer 3 minutes, then they are transferred for 8-12 minutes in 300-500 μl of balancing solution 1, which contains two cryoprotectants: EG and DMSO in equal concentrations from 5 to 10%, then from solution 1 is transferred for 1-2 minutes in 300- 500 μl of vitrification solution 2, which soda it contains two cryoprotectants EG and DMSO in concentrations from 11 to 20%, then from solution 2 is transferred for 20-40 s in 300-500 μl of solution 3, which contains three cryoprotectants EG and DMSO in concentrations from 11 to 20% and sucrose from 0 5 to 1 M, after which the contents with oocytes in a volume of 5-50 μl are transferred to a cryovial partially immersed in liquid nitrogen, the cap is tightly screwed, the cryovial is immersed in a container with liquid nitrogen and the cryovial is transferred from the container with liquid nitrogen to a Dewar vessel or cryogenic storage with liquid nitrogen. 2. The method of claim. 1, characterized in that the cow ovaries delivered to the laboratory for several hours at a temperature of 23-25 ° ^C. 3. The method according to p. 1, characterized in that the extraction of oocytes from the ovaries of cows by aspiration is carried out from antral follicles more than 2 mm in diameter. 4. The method according to p. 1, characterized in that the quick freezing of oocytes is carried out under aseptic conditions (laminar box) in order to avoid contamination. 5. The method according to p. 1, characterized in that from one to 5 oocytes can be frozen in one cryovial. 6. The method according to p. 1, characterized in that in one prepared portion of the medium can be carried out up to 5 vitrification cycles for one individual animal. 7. The method according to p. 1, characterized in that the complete procedure, including placing the oocytes in solution 3, loading into a cryovial and sealing it, should take no more than 60 s before immersion in liquid nitrogen, in order to maintain a high level of survival.

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