CA2034054C - Cryopreservation process for direct transfer of embryos - Google Patents
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Abstract
An improved cryopreservation of embryos has been developed. After freezing in a cryoprotective solution of ethylene glycol, dimethyl sulfoxide, glycerol or a combination thereof, the embryos can be thawed and transferred directly to the recipient animal without serial rehydration. The transfer can be made directly from the cryoprotective container to the recipient animal.
Description
.-- 203~E)~~
CRYOPRESERVATION PROCESS FOR
DIRECT TRANSFER OF EMBRYOS
TECHNICAL FIELD
The present invention is a process for cryopreser-vation of embryos . Af ter thawing, the embryos can be transferred directly to recipient animals without special handling. The cryoprotectant can be ethylene glycol, dimethyl sulfoxide (DMSO), glycerol, or any combination thereof. Another aspect of this invention involves pretreating the embryos with various detergents, organic solvents and proteolytic enzymes.
In addition, trophoblastic tissues from other viable embryos can be processed with the embryos to enhance the results of the cryopreservation process.
BACKGROUND
Embryos in the morula or blastocyst stage can be frozen in a cryoprotectant solution and later thawed.
The thawed embryos have a reasonably good viability rate and are transferred to the uterus of recipient animals for further gestation. The typical procedure in the embryo transfer industry requires a serial rehydration of embryos following thawing. The embryo must be removed from the freezing container, and handled by a technician. The process can take at least 30 to 40 minutes. The complicated transfer methods devised by prior investigators in this field require the concen-tration of cryoprotectant in the embryo freezing so-lution to be decreased slowly after thawing to avoid lysing embryonic cell membranes. See e-g Miyamoto, H.
and Ishibashi, T., "The Protective Action of Glycols _ 1 _.
~~34~~4 Against Freezing Damage of Mouse and Rat Embryos", J.
Reprod. Fert., 54: 427-32 (1978). This "step-wise rehydration" of the embryo has been assumed to be a necessary part of embryo handling. More recently, some investigators have rehydrated thawed embryo cells using sucrose as an osmotic buffer. Renard, J., Ozil, J., and Heyman, Y., "Cervical Transfer of Deep Frozen Cattle Embryos", Theriogenology, 15: 311-320 (1981).
In an embryo transfer technique, the primary means for cryopreservation of mammalian embryos employs the use of permeating cryoprotective agents. Glycerol is the most commonly used cryoprotectant today. Under the "step-wise rehydration" technique, embryos are most often frozen in semen straws, and upon thawing, are removed from the straws through a series of solutions containing decreasing concentrations of cryoprotectant and then reloaded into a semen straw for nonsurgical transfer to a recipient female. All embryo handling procedures require the use of a microscope, sterile supplies and solutions, and must be performed by a skilled technician trained in these procedures. There-fore, developing a process by which the frozen embryo can be thawed within the straw and transferred directly to a recipient female without seriously diminishing pregnancy rates would be very important commercially.
Currently, the most commonly employed cryoprotective agent is glycerol. Leibo, S.P. "A
One-Step Method for Direct Nonsurgical Transfer of Frozen-Thawed Bovine Embryos", Theriogenology, 21:
767-90 (1984). Among the other cryoprotectants inves-tigators have looked at using is ethylene glycol, which has been researched as a cryoprotectant for mammalian embryos, but not using a direct transfer method.
Heyman, Y. , Vincent, C. , Gamier, V. , and Cognie, Y. , "Transfer of Frozen-Thawed Embryos in Sheep", Veterinary Record, 120: 83-85 (1987). The procedure published by Y. Heyman, et al, in 1987, using ethylene glycol as a _ 2 _ cryoprotectant for sheep embryos, requires a "one-step"
dilution or rehydration with sucrose. Other investigators found ethylene glycol to be a good cryoprotectant for murine embryos, but their method still requires a three-step rehydration with a modified Krebs-Ringer bicarbonate medium. Miyamoto, H. and Ishibashi, T. "The Protective Action of Glycols Against Freezing Damage of Mouse and Rat Embryos", J. Reprod.
Fert., 54: 427-32 (1978). Miyamoto, H. and Ishibashi, T., "Survival of Frozen-Thawed Mouse and Rat Embryos in the Presence of Ethylene Glycol", J. Reprod. Fert., 50:
373-75 (1977). Other investigators have looked at direct transfer methods, but these experiments have required a combination of glycerol and sucrose as the cryoprotective agent. Massip, A., Van Der Zwalmen, P.
and Ectors, F., "Recent Progress in Cryopreservation of Cattle Embryos", Theriogenology, 27: 69-79 (1987).
One rehydration technique referred to as the "one-step" method has one rehydration step rather than a multiple step-wise rehydration. Leibo, S.P. "Embryo Transfer Method and Apparatus", U.S. Patent No.
4,380,997, issued April 26, 1983. However, even the "one-step" method involves special handling of embryos in the field because it still requires dilution of the cryoprotectant upon thawing. Although patented and described in the scientific literature, the results of this procedure "have been less than satisfactory in that only a 26$ pregnancy [rate] has been achieved overall."
Leibo, S.P. "A One-Step Method for Direct Nonsurgical Transfer of Frozen-Thawed Bovine Embryos", Theriogenology, 21: 767-90 (1984). Leibo reported highly variable results for individual sets of frozen-thawed embryos with pregnancy rates ranging from 0~ and 6~ to 55~ and 63$. It is believed that the technician performing the th~~w exerts a significant effect on pregnancy rates suggesting that this procedure is too elaborate to be reproducible enough to meet commercial needs.
SUMMARY OF THE INVENTION
The improved direct transfer method for embryonic cryopreservation and subsequent transfer to a recipient animal allows the embryos to be thawed within the cryoprotective container and successfully transferred directly from the container to the recipient animal without a rehydration process. The direct transfer method has been tested on bovine embryos and also will have future application to the embryos of other mammalian species. The present invention includes an improved method for thawing frozen embryos and transferring them at the field location of the recipient animal with consistent and successful results. The direct transfer method is a great advantage since present techniques require either a mufti-step or one-step dilution process to remove the cryoprotective agent from the embryo such that the ability of the technician performing the dilution and transfer may significantly impact pregnancy rates.
In accordance with one aspect of the invention there is provided a method for embryonic preservation for subsequent culture comprising the steps of (a) collecting embryos; (b) placing the embryos in a cryoprotective container with a cryoprotectant from a group consisting of ethylene glycol, dimethyl sulfoxide, glycerol, and any combinations thereof in a cell culture media of an isotonic salt solution; (c) allowing the embryos to equilibrate in the cryoprotective solution; (d) freezing the embryos for a desired time period; (e) thawing the embryos; and (f) directly transferring the thawed embryos.
In the present invention, embryos are collected, placed in a cryoprotective solution of from about 1.0 M to about 2.0 M ethylene glycol, DMSO, or glycerol or any combination thereof in an isotonic salt solution in containers suitable for cryopreservation that preferably allow for direct transfer to recipient animals. A preferred cryoprotective solution is about 1.5 M ethylene glycol in an isotonic salt solution. The embryos are then equilibrated and frozen for a desired length of time in liquid nitrogen. Upon thawing, the embryo in the cryoprotective container can be transferred directly to a recipient animal or to an in vitro culture system.
The present invention can also include use of embryos produced by various in vitro techniques including nuclear transfer, embryo splitting, fertilization of - 4a -..rt 2~~~a~~
in vitro matured oocytes, in vitro fertilization of oocytes, in vitro culture of fertilized oocytes, and artificial insemination. These techniques are of increasing commercial importance.
In addition, other methods of the invention include the additional step of exposing the embryos to various pretreatments of proteolytic enzymes such as pronase, trypsin, and chymotrypsin; detergents; and organic solvents such as ethanol and methanol. These pretreat-ments increase membrane permeability by either removing proteins from the cell membranes or solubilizing lipids in the cell membranes. Enhancing membrane permeability reduces osmotic shock to the embryo during equilibration of the embryo in the cryoprotective solution and during rehydration of the embryo in the recipient animal's uterus following direct transfer to the recipient.
An alternative method of the invention includes the additional step of combining the embryos with pieces of trophoblastic tissue from another viable embryo prior to equilibration. Trophoblastic tissues give rise to the placental membrane in a developing fetus. These tissues are a source of the signals which maintain pregnancy in most animals. The extra trophoblastic tissues provide signals that supplement the signals of the trophoblastic tissues of the embryo of merit, and will enhance preg-nancy rates.
The direct transfer method allows the rehydration step after thawing to be eliminated, thus allowing direct transfer of the embryo to the recipient animal.
It is no longer necessary to use a diluent such as sucrose in an isotonic salt solution in the cryo-protective container to dilute away the cryopro-tective agent in the present invention. ,Prior methods require the dilution of the cryoprotective agent, usually glycerol, from the embryo prior to transfer to the recipient animal using either a step-wise or one-step rehydration technique. The one-step technique ..~ ~.~3~~~~
which allowed for the dilution of the cryoprotectant from the embryo within the cryoprotective container required mixing of the container contents prior to transfer of the embryo to the recipient. The present invention eliminates both the dilution step and the mixing step that were required by the "one-step" method.
Furthermore, the present invention eliminates the need for a combination of glycerol and sucrose in the cryoprotective solution in order to accomplish a direct transfer such as described by Massip, et al.
BRIEF DESCRIPTION OF THE DRAWING
FIG.1 is a cross-sectional schematic view of an elongated tubular cryoprotective container (straw) with the embryo in the cryoprotective solution.
DESCRIPTION OF THE PREFERRED METHOD
The method involves several steps which generally include collecting embryos, placing the embryos in a cryoprotective solution of ethylene glycol, DMSO, glycerol, or any combination thereof in an isotonic salt solution in a cryoprotective container, allowing the embryos to equilibrate in the cryoprotectant, freezing the embryos for a desired time period in liquid nitrogen, thawing the embryos, and directly transferring the thawed embryos to the recipient animals.
The embryos are collected from artificially inseminated animals as well as created by in vitro procedures, including in vitro fertilization of in vivo and in vitro matured oocytes, in vitro culture of fertilized oocytes, embryo splitting, and nuclear transfer. Any method to produce properly aged embryos may be used.
The embryo splitting technique is well-known to those skilled in the art. One preferred method of embryo splitting involves cutting normal 6 to 8 Day embryos into two pieces with a micromanipulator.
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Monozygotic twins are thereby induced. The other methods of producing embryos are discussed in more detail subsequently.
The embryos are placed in a cryoprotective con-tainer with a cryoprotectant mixed with a cell culture media of an isotonic salt solution. The isotonic salt solution generally has an osmolality of 250 to 350 milliosmoles, a pH in the range of 6.8 to 7.6, a pH
buffer, and is supplemented with a protein source or a synthetic macromolecule such as polyvinylpyrrolidone.
The solution may contain individual nutrients such as carbohydrates, amino acids and vitamins. The most common salt solution used for freezing embryos is Dulbecco' s phosphate buffered saline with common modi-fications of 0.4$ bovine serum albumin, 0.1~ glucose, and 0.036 sodium pyruvate (the solution hereinafter referred to as PBl).
A cryoprotective solution using one cryoprotectant is in the range of about 1 M to about 2 M. When the cryoprotective solution is a combination of cryoprotectants from the group ethylene glycol, DMSO, and glycerol, the primary cryoprotectant in the solution will be ethylene glycol supplemented with a lower concentration of one of the other cryoprotectants listed. In one preferred method, ethylene glycol will be in the range of about 1.0 M to 1.25 M, and the second cryoprotectant, that is combined with the ethylene glycol, will be in the range of about 0.5 M to 0.25 M.
The combination of cryoprotectants will be in the isotonic salt solution that was already discussed.
An alternative method of the invention includes the additional step of combining the embryos with pieces of trophoblastic tissue from different viable embryos prior to equilibration of the embryo in the cryoprotective solution. As discussed before, trophoblastic tissues give rise to the placental membranes in a developing fetus and are a source of the signals which maintain PATENT
pregnancy in most animals. This is generally known to those well-skilled ir. the art.
In one preferred method, the trophoblastic tissue can be freshly dissected from a viable embryo at about Day 13 to Day 14 or it can be dissected and cultured for twenty-four hours in a suitable culture medium such as tissue culture medium (TCM) 199 supplemented with 10$
fetal calf serum (FCS). The trophoblastic tissue is combined with the embryo in the cryoprotective solution and otherwise processed according to the methods of this invention. The trophoblastic tissue and embryo there-fore can be directly transferred from the cryoprotective container to the uterus of the recipient animal.
After the embryos are placed in the cryoprotective solution they are allowed to equilibrate for about 5 to 40 minutes in the temperature range of above 0°C to 39°C. Preferably the equilibration time is in the range of about 10 to about 20 minutes, and the equilibration temperature is in the range of about 18°C to 25°C. In general, the appropriate equilibration time will depend on the environmental conditions which the embryos are subjected to; i.e., the concentration of cryoprotectant and the temperature at which equilibration is performed.
Generally, as the cryoprotectant concentration increases, the time required for equilibration decreases, and vice versa. Furthermore, as the temperature increases, the equilibration time required decreases. The term "equilibrate" as used herein means the interior of the embryo is reaching a condition of relative balance with the cryoprotective solution.
The placement of the embryo in the cryoprotective solution takes place in a petri dish with subsequent equilibration preferably taking place in a container suitable for cryopreservation that allows for direct transfer to recipient animals. The preferred embodiment is a cryoprotective container in a tubular shape and made of a biocompatible plastic that can be inserted _ g _ .....
PATENT
into a standard artificial insemination gun or an artificial insemination gun modified for embryo transfer (hereinafter referred to as an artificial insemination gun). This allows the embryo to remain in the container throughout freezing, thawing, and transfer procedures thereby eliminating handling of the embryo.
One preferred process of the invention employs the use of sterile plastic semen straws commonly used in the artificial insemination industry. Such straws are familiar to those skilled in the art. The straws are available in a variety of sizes, and the 0.25m1 and 0.50 ml capacity straws are suitable for the present in-vention.
Referring to FIG. 1, one preferred embodiment of the cryoprotective container is shown after the embryo has been prepared for freezing. FIG. 1 is a cross-section of a tubular container 1, which represents a suitable structure, such as the plastic semen straws previously mentioned. Figure 1 depicts the tubular container 1 after the embryo has been placed inside and sealed. The tubular container 1 is closed at one end 2 by a layer of porous sealing plug material 3, which could be cotton, a sealant 4 above that, and another layer of porous sealing plug material 5 above that. The preferred sealant 4 is a dry powder material such as polyvinyl alcohol which solidifies and seals once it becomes moist. The tubular container 1 has an open end opposite to end 2 for receiving the appropriate volumes of liquid and embryo. The first column of liquid 6 above the plug material 5 contains isotonic salt solution or the cryoprotective solution. Above the first column of liquid is a second column of liquid 9 which is separated from the first by an air bubble 8.
The second column of liquid 9 is a cryoprotective solution which contains the embryo 10. An air bubble 12 separates the second column of liquid 9 from a third column of liquid 13. The third column of liquid may be _ g _ ~~~~Qa4 PATENT
isotonic salt solution or cryoprotective solution.
Typically, the third or top column 13 contains the isotonic salt solution. The overall ratio of the isotonic salt solution to the cryoprotective solution in the container is in the range of about 3:1 to 4:1. The upper end of the tubular container is heat sealed by melting the end of the plastic straw to form a seal 15.
The solutions in the third column 13 and first column 6 of the container help to assure that the embryo is expelled from the gun when transferred to the recipient.
One preferred technique for loading the container shown in FIG.1 is to use a syringe to first draw the isotonic salt solution into the container to form the first column 6, then to allow the container to aspirate air bubble 8. A syringe is used to introduce the volume 9 of cryoprotective agent containing the embryo 10 into the container. Next, the container should be allowed to aspirate another air bubble 12, and finally the isotonic salt solution or the cryoprotective solution 13 should be drawn into the container with the syringe. Continued aspiration with the syringe will cause the isotonic salt solution in column 6 to contact layers 5 and 4 so that the sealant 4 solidifies. The container can then be heat sealed.
When the cryoprotective container is ready to be loaded into the artificial insemination gun, the heat sealed end 15 can be cut off and then the tubular container can be placed into the artificial insemination gun. The artificial insemination gun pushes the sealing plug 4 through the interior diameter of the tubular container forcing all of the liquid material in the container out its open end directly into the recipient animal.
Freezing the embryos involves first cooling them in a controlled rate freezer. The embryos can be either placed directly into the freezing apparatus at a temper-ature slightly below the freezing point of the PATENT
cryoprotective solution, which is called the seeding temperature, or can be cooled at a controlled rate of about -1°C/minute to the seeding temperature. The seeding temperature is normally in the range of about -5°C to -8°C. The embryos are held at this seeding temperature long enough to reach thermal equilibrium between the container and the freezer chamber which usually takes from about 1 to about 2 minutes. Ice nucleation is then induced by touching the container with a metal instrument previously supercooled in liquid nitrogen. The embryos are then cooled further at a controlled rate in the range of about -0.1°C/minute to about -1.0°C/minute until the embryos are in the range of about -2 0 ° C to -40 ° C . One pref erred method of the invention is to further cool the embryos after ice nucleation at the controlled rate of -0.3°C/minute to -0.5°C/minute to an end point temperature in the range of about -25°C to about -35°C. Once the embryos reach the end point temperature, they can be plunged immediately into liquid nitrogen or held at the endpoint temperature for a varying period of time prior to plunging, the preferred length of the hold being 15 minutes.
When the time comes to remove the embryos from storage in liquid nitrogen for transfer to recipient animals, the thawing procedure chosen should be suitable for the freezing procedures employed; i.e., the insulat-ing properties of the container, the cooling rates used and the terminal temperature at which the embryo is plunged into liquid nitrogen. It_is generally accepted that when an embryo is slow cooled to an endpoint temperature below -40°C (usually to -60°C to -80°C) before plunging the embryo into liquid nitrogen, a slow thaw rate (relative to the thaw rate required if the embryo is plunged into liquid nitrogen at a temperature greater than -40°C) is required to maintain embryo viability.
The thawing procedures for embryos plunged in liquid nitrogen at temperatures greater than -40°C may range from placing the cryoprotective container in air at room temperature to placing the container in a water bath of up to 40°C. In the preferred method of this invention the cryoprotective containers are placed into a water bath of about 30°C for about seconds.
As discussed before, the thawed embryo is then directly transferred to the recipient animal. In the preferred 10 method, this transfer is done immediately upon removal of the container from the water bath. Generally, this is done by loading the cryoprotective container into an artificial insemination gun and using the gun to deposit the embryo into the uterus of a suitable recipient female. Furthermore, the direct transfer step may include transferring the embryo from the cryoprotective container to an in vitro culture system and then, at a desired stage in its development, transferring the embryo to a recipient animal. The ultimate recipient is an animal of the same species as the embryo. An intermediate gestation may involve a suitable surrogate of another species.
In a preferred embodiment the invention provides a method for embryonic preservation of a bovine embryo or an embryo of another mammalian species, comprising a method for embryonic preservation for subsequent culture comprising the steps of: collecting Day 7 to 7 1/2 embryos in the late morula to expanded blastocyst stage; placing the embryos in a cryoprotective solution of from about 1.0 M to about 2.0 M
ethylene glycol and PB1 in plastic semen straws; allowing the embryos to equilibrate for about 10 minutes at room temperature; cooling the embryos in an alcohol bath freezer at -7°C for 2 minutes; inducing ice nucleation by touching the straw with a supercooled metal instrument; holding the embryos for 5 minutes at -7°C; cooling at a controlled rate of -0.5°C/minute to a final temperature of -35°C; holding at -35°C
for 15 minutes; plunging the embryos into liquid nitrogen for long term storage; thawing the embryos by placing the straws into a water bath of about 30°C for 10 seconds; and transferring the thawed embryos directly to a recipient mammalian female using an artificial insemination gun suitable for embryo transfer.
The following examples are provided to facilitate the understanding of preferred methods of the present invention and not for the purpose of limiting same. Those skilled in the art will recognize that various modifications in the procedure previously discussed and outlined below can be used for the purpose of practicing the present invention.
Bovine embryos can be collected in the range of Day 6 to Day 8 of the donor estrous cycle in the morula to hatched blastocyst stage, but in the preferred method are collected at Day 7 to Day 7 1/2 in the late morula to expanded blastocyst stage, placed in a cryoprotective solution of 1.5 M ethylene glycol in PB1, and loaded - 12a -PATENT
into a 0.25 ml plastic semen straw with a section of a 0.5 ml semen straw fitted over the sealed end for use as a handle and a labelling surface (Continental Plastics or I.M.V.). Next the embryos are allowed to equilibrate for ten minutes at room temperature. The freezing procedure for the embryos involves cooling the embryos in a controlled rate freezer to -7°C for two minutes, inducing ice nucleation by touching the straw with a supercooled metal instrument such as metal forceps, holding the embryos for 5 minutes at -7°C, cooling the embryos at a controlled rate of -0.5°C/minute to an end point temperature of -35°C, holding the embryos at -35°C
for 15 minutes, and then plunging the embryos into liquid nitrogen for final storage.
At the appropriate time for transfer of the embryos to the recipient animal, the embryos are thawed by placing the semen straws into a water bath of about 30°C
for ten seconds. The top end of the straw is then snipped off just below the heat seal and the straw is inserted into an artificial insemination gun. The entire contents of the straw are transferred to the uterus of an appropriate recipient animal by causing the plunger of the artificial insemination gun to push the plug at the bottom end of the straw through the length of the straw delivering the liquid contents and embryo out the open cut off end of the straw into the uterus of the animal.
Tables 1 and 2 show the results of this preferred method of the invention. The two tables complement each other in that Table 1 shows viability rates of the embryos after direct transfer to an in vitro culture, while Table 2 shows pregnancy rates resulting from direct transfer to a recipient cow. In these tables and the subsequent tables, the resul..ts of the preferred method are compared to a control of 10~ glycerol using a three-step rehydration procedure unless otherwise specified. This control was chosen because of the PATENT
widespread use of glycerol and step-wise rehydration methods in the cryoprotective field.
Table 1 below shows bovine embryo viability at 24, 48 and 72 hours for embryos in a culture medium with the number of viable embryos compared to the number of embryos frozen and the percentage of viable embryos listed in parentheses. Embryos frozen in glycerol were rehydrated using a step-wise procedure. Embryos frozen in 1.5 t4 ethylene glycol were transferred directly into tissue culture media (TCM) 199 with 10$ FCS.
TABLE 1. IN VITRO DEVELOPMENT OF EMBRYOS FROZEN
IN ETHYLENE GLYCOL AND REHYDRATED DIRECTLY
No. viable/No, frozen ($) Cryoprotectant 24 hours 48 hours 72 hours 10~ Glycerol 6/7 (86) 6/7 (86) 6/7 (86) 1.5 M Eth. Gly. 24/25 (96) 23/25 (96) 22/25 (88) These results demonstrate that embryos have a high survival rate with direct rehydration when frozen in 1.5 M ethylene glycol.
Table 2 below shows the number of bovine pregnancies compared to the number of embryos transferred to recipients for straws loaded with different solutions. The first column of data is for a straw loaded with 1.5 M ethylene glycol, in all three columns, (columns 6, 9 and 13 of Figure 1), while the second column of data shows pregnancy rates for a straw loaded with 1.5 M ethylene glycol in columns 9 and 13 of Figure 1 and PB1 in column 6 with the ratio of ethylene glycol to PB1 being 1:3. The third column of data is PATENT
for a straw loaded with 10~ glycerol in all three columns. Embryos frozen in glycerol were rehydrated using a three-step procedure. All others were transferred directly following thawing.
TABLE 2. PREGNANCY RATES AT ABOUT
No. pregnant/No. transferred within the indicated straw solutions Replicate 1.5 M Eth.Gly. 1.5 M Eth.Gly/PB1 10~ Glycerol Total 10/26 12/24 20/35 (38$) (50~) (57~) The results in Table 1 indicate higher viability for embryos in 1.5 M ethylene glycol rather than 10$
glycerol after direct transfer to an in vitro culture.
The data on pregnancy rates in Table 2 shows similar results for 1.5 M ethylene glycol and 10$ glycerol, especially when the straw is loaded with ethylene glycol and the PB1 in a 1:3 ratio.
Of the 22 pregnancies resulting from the direct transfers shown in Table 2, two have been allowed to proceed as normal pregnancies, and live calves are expected on about June 24, 1990. The other 20 preg-nancies were proceeding normally at greater than 40 days of gestation and were purposely aborted in order to reuse the recipient females in subsequent trials.
PATENT
The following is an alternative method using a cryopreservation pretreatment of trypsin.
This method is accomplished by following the steps of Example 1 with the additional step of exposing the embryos to a solution containing trypsin in the range of about 0.05 to 0.5~ in an isotonic salt solution prior to equilibrating the embryos in the cryoprotective solution. The embryos are washed in trypsin using a procedure similar to the one recommended by the Interna-tional Embryo Transfer Society (IETS). This procedure involves washing the embryos five times in Dulbecco's phosphate buffered saline with 0.4~ bovine serum albumin (BSA), washing the embryos two times (60 to 90 seconds each) in 0.25 trypsin in calcium and magnesium-free Hank's balanced salt solution (HBSS), and then washing the embryos five times in calcium and magnesium free Dulbecco's phosphate buffered saline with 10$ fetal calf serum (FCS).
Table 3 shows the effect of trypsin washing on the in vitro development of embryos frozen in ethylene glycol and rehydrated directly in a culture medium. In each column of data the number of viable bovine embryos at 24, 48 and 72 hours is compared to the number thawed and rehydrated directly. The percentage of viable embryos is shown in parentheses.
?(13~0~4 PATENT
TABLE 3. EFFECT OF TRYPSIN WASHING ON IN VITRO
DEVELOPMENT OF EMBRYOS FROZEN
IN ETHYLENE GLYCOL AND REHYDRATED DIRECTLY
No. viable/No. thawed (~) Treatment 24 hours 48 hours 72 hours Trypsin washed 1.5 M ethylene glycol Rep I 14/19 (74) 14/19 (74) 12/19 (63) Rep II 16/21 (76) 16/21 (76) 16/21 (76) Total 30/40 (75) 30/40 (75) 28/40 (70) Control washed 1.5 M ethylene glycol Rep I 13/19 (68) 13/19 (68) 8/19 (42) Rep II 19/21 (90) 19/21 (90) 18/21 (86) Total 32/40 (80) 32/40 (80) 26/40 (65) Embryos in replicate I were generally of poor quality which may explain their poor development compared with other ethylene glycol experiments.
Replicate II embryos were 7.5 to 8.0 day blastocysts.
Looking at replicate II, better results are achieved without a trypsin wash, but the viability rates are good for both trypsin washed and control washed embryos. The total figures indicate better results when the embryos are trypsin washed.
The following example is one preferred embodiment of the present invention in which the steps of Example 1 are followed and the direct transfer step includes the additional step of transferring the embryo from the cryoprotective container to an in vitro culture system -~ 2~3~~~4 PATENT
of PBl for a desired time period prior to transferring to the recipient animal. After transfer from the straw to PB1 the embryo can be briefly observed to assess viability and then loaded into another container for transfer to a recipient or held in culture for an extended period of time prior to transfer. In the case of the latter, the culture system would preferably consist of a bicarbonate buffered medium generally accepted for use in long-term culture of embryos and with or without feeder cells. Another option would be to transfer the embryos to the oviduct of an intermedi-ate host for temporary culture prior to transfer to a final recipient.
The following example illustrates varying concen-trations of the preferred cryoprotectant, ethylene glycol. The steps of Example 1 were followed. The cryoprotectant is ethylene glycol in the range of about 1.0 M to about 2.0 M in PB1.
Table 4 below shows bovine embryo viability at 24, 48 and 72 hours for different concentrations of ethylene glycol in the cryoprotective solution.
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PATENT
TABLE 4. IN VITRO DEVELOPMENT OF EMBRYOS FROZEN IN
1.0 M, 1.5 M, AND 2.0 M ETHYLENE GLYCOL
AND REHYDRATED DIRECTLY
No. viable/No. frozen Cryoprotectant 24 hours 48 hours 72 hours 10~ Glycerol 5/8 (63) 5/8 (63) 5/8 (63) 1.0 M Eth.Gly. 14/20 (70) 12/20 (60) 11/20 (55) 1.5 M Eth.Gly. 17/20 (85) 17/20 (85) 16/20 (80) 2.0 M Eth.Gly. 8/25 (32) 12/25 (48) 8/25 (32) The preferred concentration is about 1.5 M ethylene glycol based on this example's data. Embryos frozen in the glycerol control were rehydrated using a step-wise procedure.
Another alternative method of the present invention is shown in the following example. The steps of Example 1 are followed using different cryoprotective solutions of 10~ glycerol, 1.5 M propylene glycol in PB1 and 1.5 M
DMSO in PB1.
Table 5 below shows bovine embryo viability at 24, 48 and 72 hours for these cryoprotective solutions.
TABLE 5. IN VITRO DEVELOPMENT OF
- EMBRYOS FROZELJ IN GLYCEROL, ETHYLEPJE GLYCOL, PROPYLENE GLYCOL OR DMSO AND REHYDRATED DIRECTLY
No. (~) viable Cryoprotectant 24 hours 48 hours 72 hours 10'~ Glycerol 6/10 (60) 3/10 (30) 3/10 (30) 1.5 M Eth.Gly. 16/20 (80) 15/20 (75) 14/20 (70) 1.5 M DMSO 7/20 (35) 7/20 (35) 5/20 (35) 1.5 M Prop.Gly. 3/19 (16) 3/19 (i6) 2/19 (11) All embryos were placed directly into holding media after thawing. This experiment showed that the direct rehydration procedure does work with other cryoprotectants but not as well as with ethylene glycol.
The embryos used for the direct transfer technique can be developed using nuclear transfer techniques. The donor cell, typically an embryonic cell, is fused to an enucleated egg thereby transferring the nuclear material. A nuclear transfer procedure for bovine embryos is described in Bovine Nuclear Transplantation, Massey and Willadsen, PCT published application WO
88/09816, 15 Dec. 1988. -Those skilled in the art are familiar with other nuclear transfer procedures.
Table 6 shows in vitro development of cloned embryos produced by nuclear transfer frozen in 10~
glycerol and rehydrated using the step-wise procedure compared to cloned embryos produced by nuclear transfer, ~o~~o~~
PATENT
frozen in 1.5 M ethylene glycol, thawed, and rehydrated directly in the culture medium. Embryo viability is shown at 24, 48 and 72 hours.
TABLE 6. IN VITRO DEVELOPMENT OF EMBRYOS
PRODUCED BY NUCLEAR TRANSFER, FROZEN IN ETHYLENE GLYCOL, AND REHYDRATED DIRECTLY
No. ($) viable Cryoprotectant 24 hours 48 hours 72 hours 10$ Glycerol 14/27 (52) 13/27 (48) 13/27 (48) 1.5 M Eth.Gly. 18/28 (64) 14/28 (50) 14/28 (50) Embryos frozen in glycerol were rehydrated using a step-wise procedure. Embryos frozen in 1.5 M ethylene glycol were placed directly into a cell culture media of tissue culture media (TCM) 199 with 10$ FCS. The data shows that the direct transfer procedure is successful on nuclear transfer embryos.
Table 7 shows the pregnancies achieved in a first trial of direct transfer of cloned embryos produced by nuclear transfer to recipient females.
TABLE 7. PREGNANCY RATES OF DIRECT TRANSFER TRIALS
USING NUCLEAR TRANSFER EMBRYOS
Cryoprotectant- No. regnant/No transferred (~) 10~ Glycerol 1/11 (9) 1.5 M Ethylene Glycol 3/11 (27) Embryos frozen in glycerol were rehydrated using a step-wise procedure. All others were transferred directly following thawing. This data illustrates that the direct transfer procedure has value for cryopreser-vation of nuclear transfer embryos. The three recipient females pregnant with calves produced from direct transfer of nuclear transfer embryos are presently pregnant and will be maintained until parturition in about August and September of 1990. Therefore, a total of 5 pregnancies from the use of direct transfer proce-dures are presently being maintained.
The embryos used for the direct transfer technique can be developed using embryos that are cultured in vitro for part or all of the time to the desired age for cryopreservation. The fertilized eggs are placed in the appropriate culture medium for development. Many techniques and culture media are.known in the art. A
method for bovine embryo development is described in Bovine Embryo In Vitro Culture, Bondioli, PCT published Application WO 89/07135, 10 Aug. 1989.
References to other embryo culture systems are included in WO 89/07135 and are exemplary of alternative methods.
~--PATENT
The direct transfer method of this invention can be used with embryos that have been developed from an in vitro matured oocyte. The immature oocytes are collect-ed and cultured prior to fertilization. Various tech-niques have been known to those skilled in the art as described in Edwards, "Maturation In Vitro of Mouse, Sheep, Cow, Pig, Rhesus Monkey and Human Ovarian Oocytes," Nature, Vol. 208, pp. 349-351 (1965). More recently culture systems have been developed for various mammalian species including bovine oocytes. See e-g., Lu et al., "Pregnancy Established in Cattle by Transfer of Embryos Derived from In Vitro Fertilization of Oocytes Matured In Vitro," Vet. Rec., Vol. 121, pp.
259-260 (1987). Any successful in vitro oocyte matura-tion process can be used to produce the embryos to be frozen and subsequently transferred according to the method of this invention.
Those skilled in the art upon reading the above detailed description of the present invention will appreciate that many modifications of the method de-scribed above can be made without departing from the spirit of the invention. All such modifications which fall within the scope of the appended claims are intend-ed to be covered thereby.
CRYOPRESERVATION PROCESS FOR
DIRECT TRANSFER OF EMBRYOS
TECHNICAL FIELD
The present invention is a process for cryopreser-vation of embryos . Af ter thawing, the embryos can be transferred directly to recipient animals without special handling. The cryoprotectant can be ethylene glycol, dimethyl sulfoxide (DMSO), glycerol, or any combination thereof. Another aspect of this invention involves pretreating the embryos with various detergents, organic solvents and proteolytic enzymes.
In addition, trophoblastic tissues from other viable embryos can be processed with the embryos to enhance the results of the cryopreservation process.
BACKGROUND
Embryos in the morula or blastocyst stage can be frozen in a cryoprotectant solution and later thawed.
The thawed embryos have a reasonably good viability rate and are transferred to the uterus of recipient animals for further gestation. The typical procedure in the embryo transfer industry requires a serial rehydration of embryos following thawing. The embryo must be removed from the freezing container, and handled by a technician. The process can take at least 30 to 40 minutes. The complicated transfer methods devised by prior investigators in this field require the concen-tration of cryoprotectant in the embryo freezing so-lution to be decreased slowly after thawing to avoid lysing embryonic cell membranes. See e-g Miyamoto, H.
and Ishibashi, T., "The Protective Action of Glycols _ 1 _.
~~34~~4 Against Freezing Damage of Mouse and Rat Embryos", J.
Reprod. Fert., 54: 427-32 (1978). This "step-wise rehydration" of the embryo has been assumed to be a necessary part of embryo handling. More recently, some investigators have rehydrated thawed embryo cells using sucrose as an osmotic buffer. Renard, J., Ozil, J., and Heyman, Y., "Cervical Transfer of Deep Frozen Cattle Embryos", Theriogenology, 15: 311-320 (1981).
In an embryo transfer technique, the primary means for cryopreservation of mammalian embryos employs the use of permeating cryoprotective agents. Glycerol is the most commonly used cryoprotectant today. Under the "step-wise rehydration" technique, embryos are most often frozen in semen straws, and upon thawing, are removed from the straws through a series of solutions containing decreasing concentrations of cryoprotectant and then reloaded into a semen straw for nonsurgical transfer to a recipient female. All embryo handling procedures require the use of a microscope, sterile supplies and solutions, and must be performed by a skilled technician trained in these procedures. There-fore, developing a process by which the frozen embryo can be thawed within the straw and transferred directly to a recipient female without seriously diminishing pregnancy rates would be very important commercially.
Currently, the most commonly employed cryoprotective agent is glycerol. Leibo, S.P. "A
One-Step Method for Direct Nonsurgical Transfer of Frozen-Thawed Bovine Embryos", Theriogenology, 21:
767-90 (1984). Among the other cryoprotectants inves-tigators have looked at using is ethylene glycol, which has been researched as a cryoprotectant for mammalian embryos, but not using a direct transfer method.
Heyman, Y. , Vincent, C. , Gamier, V. , and Cognie, Y. , "Transfer of Frozen-Thawed Embryos in Sheep", Veterinary Record, 120: 83-85 (1987). The procedure published by Y. Heyman, et al, in 1987, using ethylene glycol as a _ 2 _ cryoprotectant for sheep embryos, requires a "one-step"
dilution or rehydration with sucrose. Other investigators found ethylene glycol to be a good cryoprotectant for murine embryos, but their method still requires a three-step rehydration with a modified Krebs-Ringer bicarbonate medium. Miyamoto, H. and Ishibashi, T. "The Protective Action of Glycols Against Freezing Damage of Mouse and Rat Embryos", J. Reprod.
Fert., 54: 427-32 (1978). Miyamoto, H. and Ishibashi, T., "Survival of Frozen-Thawed Mouse and Rat Embryos in the Presence of Ethylene Glycol", J. Reprod. Fert., 50:
373-75 (1977). Other investigators have looked at direct transfer methods, but these experiments have required a combination of glycerol and sucrose as the cryoprotective agent. Massip, A., Van Der Zwalmen, P.
and Ectors, F., "Recent Progress in Cryopreservation of Cattle Embryos", Theriogenology, 27: 69-79 (1987).
One rehydration technique referred to as the "one-step" method has one rehydration step rather than a multiple step-wise rehydration. Leibo, S.P. "Embryo Transfer Method and Apparatus", U.S. Patent No.
4,380,997, issued April 26, 1983. However, even the "one-step" method involves special handling of embryos in the field because it still requires dilution of the cryoprotectant upon thawing. Although patented and described in the scientific literature, the results of this procedure "have been less than satisfactory in that only a 26$ pregnancy [rate] has been achieved overall."
Leibo, S.P. "A One-Step Method for Direct Nonsurgical Transfer of Frozen-Thawed Bovine Embryos", Theriogenology, 21: 767-90 (1984). Leibo reported highly variable results for individual sets of frozen-thawed embryos with pregnancy rates ranging from 0~ and 6~ to 55~ and 63$. It is believed that the technician performing the th~~w exerts a significant effect on pregnancy rates suggesting that this procedure is too elaborate to be reproducible enough to meet commercial needs.
SUMMARY OF THE INVENTION
The improved direct transfer method for embryonic cryopreservation and subsequent transfer to a recipient animal allows the embryos to be thawed within the cryoprotective container and successfully transferred directly from the container to the recipient animal without a rehydration process. The direct transfer method has been tested on bovine embryos and also will have future application to the embryos of other mammalian species. The present invention includes an improved method for thawing frozen embryos and transferring them at the field location of the recipient animal with consistent and successful results. The direct transfer method is a great advantage since present techniques require either a mufti-step or one-step dilution process to remove the cryoprotective agent from the embryo such that the ability of the technician performing the dilution and transfer may significantly impact pregnancy rates.
In accordance with one aspect of the invention there is provided a method for embryonic preservation for subsequent culture comprising the steps of (a) collecting embryos; (b) placing the embryos in a cryoprotective container with a cryoprotectant from a group consisting of ethylene glycol, dimethyl sulfoxide, glycerol, and any combinations thereof in a cell culture media of an isotonic salt solution; (c) allowing the embryos to equilibrate in the cryoprotective solution; (d) freezing the embryos for a desired time period; (e) thawing the embryos; and (f) directly transferring the thawed embryos.
In the present invention, embryos are collected, placed in a cryoprotective solution of from about 1.0 M to about 2.0 M ethylene glycol, DMSO, or glycerol or any combination thereof in an isotonic salt solution in containers suitable for cryopreservation that preferably allow for direct transfer to recipient animals. A preferred cryoprotective solution is about 1.5 M ethylene glycol in an isotonic salt solution. The embryos are then equilibrated and frozen for a desired length of time in liquid nitrogen. Upon thawing, the embryo in the cryoprotective container can be transferred directly to a recipient animal or to an in vitro culture system.
The present invention can also include use of embryos produced by various in vitro techniques including nuclear transfer, embryo splitting, fertilization of - 4a -..rt 2~~~a~~
in vitro matured oocytes, in vitro fertilization of oocytes, in vitro culture of fertilized oocytes, and artificial insemination. These techniques are of increasing commercial importance.
In addition, other methods of the invention include the additional step of exposing the embryos to various pretreatments of proteolytic enzymes such as pronase, trypsin, and chymotrypsin; detergents; and organic solvents such as ethanol and methanol. These pretreat-ments increase membrane permeability by either removing proteins from the cell membranes or solubilizing lipids in the cell membranes. Enhancing membrane permeability reduces osmotic shock to the embryo during equilibration of the embryo in the cryoprotective solution and during rehydration of the embryo in the recipient animal's uterus following direct transfer to the recipient.
An alternative method of the invention includes the additional step of combining the embryos with pieces of trophoblastic tissue from another viable embryo prior to equilibration. Trophoblastic tissues give rise to the placental membrane in a developing fetus. These tissues are a source of the signals which maintain pregnancy in most animals. The extra trophoblastic tissues provide signals that supplement the signals of the trophoblastic tissues of the embryo of merit, and will enhance preg-nancy rates.
The direct transfer method allows the rehydration step after thawing to be eliminated, thus allowing direct transfer of the embryo to the recipient animal.
It is no longer necessary to use a diluent such as sucrose in an isotonic salt solution in the cryo-protective container to dilute away the cryopro-tective agent in the present invention. ,Prior methods require the dilution of the cryoprotective agent, usually glycerol, from the embryo prior to transfer to the recipient animal using either a step-wise or one-step rehydration technique. The one-step technique ..~ ~.~3~~~~
which allowed for the dilution of the cryoprotectant from the embryo within the cryoprotective container required mixing of the container contents prior to transfer of the embryo to the recipient. The present invention eliminates both the dilution step and the mixing step that were required by the "one-step" method.
Furthermore, the present invention eliminates the need for a combination of glycerol and sucrose in the cryoprotective solution in order to accomplish a direct transfer such as described by Massip, et al.
BRIEF DESCRIPTION OF THE DRAWING
FIG.1 is a cross-sectional schematic view of an elongated tubular cryoprotective container (straw) with the embryo in the cryoprotective solution.
DESCRIPTION OF THE PREFERRED METHOD
The method involves several steps which generally include collecting embryos, placing the embryos in a cryoprotective solution of ethylene glycol, DMSO, glycerol, or any combination thereof in an isotonic salt solution in a cryoprotective container, allowing the embryos to equilibrate in the cryoprotectant, freezing the embryos for a desired time period in liquid nitrogen, thawing the embryos, and directly transferring the thawed embryos to the recipient animals.
The embryos are collected from artificially inseminated animals as well as created by in vitro procedures, including in vitro fertilization of in vivo and in vitro matured oocytes, in vitro culture of fertilized oocytes, embryo splitting, and nuclear transfer. Any method to produce properly aged embryos may be used.
The embryo splitting technique is well-known to those skilled in the art. One preferred method of embryo splitting involves cutting normal 6 to 8 Day embryos into two pieces with a micromanipulator.
~~~~Q~~
Monozygotic twins are thereby induced. The other methods of producing embryos are discussed in more detail subsequently.
The embryos are placed in a cryoprotective con-tainer with a cryoprotectant mixed with a cell culture media of an isotonic salt solution. The isotonic salt solution generally has an osmolality of 250 to 350 milliosmoles, a pH in the range of 6.8 to 7.6, a pH
buffer, and is supplemented with a protein source or a synthetic macromolecule such as polyvinylpyrrolidone.
The solution may contain individual nutrients such as carbohydrates, amino acids and vitamins. The most common salt solution used for freezing embryos is Dulbecco' s phosphate buffered saline with common modi-fications of 0.4$ bovine serum albumin, 0.1~ glucose, and 0.036 sodium pyruvate (the solution hereinafter referred to as PBl).
A cryoprotective solution using one cryoprotectant is in the range of about 1 M to about 2 M. When the cryoprotective solution is a combination of cryoprotectants from the group ethylene glycol, DMSO, and glycerol, the primary cryoprotectant in the solution will be ethylene glycol supplemented with a lower concentration of one of the other cryoprotectants listed. In one preferred method, ethylene glycol will be in the range of about 1.0 M to 1.25 M, and the second cryoprotectant, that is combined with the ethylene glycol, will be in the range of about 0.5 M to 0.25 M.
The combination of cryoprotectants will be in the isotonic salt solution that was already discussed.
An alternative method of the invention includes the additional step of combining the embryos with pieces of trophoblastic tissue from different viable embryos prior to equilibration of the embryo in the cryoprotective solution. As discussed before, trophoblastic tissues give rise to the placental membranes in a developing fetus and are a source of the signals which maintain PATENT
pregnancy in most animals. This is generally known to those well-skilled ir. the art.
In one preferred method, the trophoblastic tissue can be freshly dissected from a viable embryo at about Day 13 to Day 14 or it can be dissected and cultured for twenty-four hours in a suitable culture medium such as tissue culture medium (TCM) 199 supplemented with 10$
fetal calf serum (FCS). The trophoblastic tissue is combined with the embryo in the cryoprotective solution and otherwise processed according to the methods of this invention. The trophoblastic tissue and embryo there-fore can be directly transferred from the cryoprotective container to the uterus of the recipient animal.
After the embryos are placed in the cryoprotective solution they are allowed to equilibrate for about 5 to 40 minutes in the temperature range of above 0°C to 39°C. Preferably the equilibration time is in the range of about 10 to about 20 minutes, and the equilibration temperature is in the range of about 18°C to 25°C. In general, the appropriate equilibration time will depend on the environmental conditions which the embryos are subjected to; i.e., the concentration of cryoprotectant and the temperature at which equilibration is performed.
Generally, as the cryoprotectant concentration increases, the time required for equilibration decreases, and vice versa. Furthermore, as the temperature increases, the equilibration time required decreases. The term "equilibrate" as used herein means the interior of the embryo is reaching a condition of relative balance with the cryoprotective solution.
The placement of the embryo in the cryoprotective solution takes place in a petri dish with subsequent equilibration preferably taking place in a container suitable for cryopreservation that allows for direct transfer to recipient animals. The preferred embodiment is a cryoprotective container in a tubular shape and made of a biocompatible plastic that can be inserted _ g _ .....
PATENT
into a standard artificial insemination gun or an artificial insemination gun modified for embryo transfer (hereinafter referred to as an artificial insemination gun). This allows the embryo to remain in the container throughout freezing, thawing, and transfer procedures thereby eliminating handling of the embryo.
One preferred process of the invention employs the use of sterile plastic semen straws commonly used in the artificial insemination industry. Such straws are familiar to those skilled in the art. The straws are available in a variety of sizes, and the 0.25m1 and 0.50 ml capacity straws are suitable for the present in-vention.
Referring to FIG. 1, one preferred embodiment of the cryoprotective container is shown after the embryo has been prepared for freezing. FIG. 1 is a cross-section of a tubular container 1, which represents a suitable structure, such as the plastic semen straws previously mentioned. Figure 1 depicts the tubular container 1 after the embryo has been placed inside and sealed. The tubular container 1 is closed at one end 2 by a layer of porous sealing plug material 3, which could be cotton, a sealant 4 above that, and another layer of porous sealing plug material 5 above that. The preferred sealant 4 is a dry powder material such as polyvinyl alcohol which solidifies and seals once it becomes moist. The tubular container 1 has an open end opposite to end 2 for receiving the appropriate volumes of liquid and embryo. The first column of liquid 6 above the plug material 5 contains isotonic salt solution or the cryoprotective solution. Above the first column of liquid is a second column of liquid 9 which is separated from the first by an air bubble 8.
The second column of liquid 9 is a cryoprotective solution which contains the embryo 10. An air bubble 12 separates the second column of liquid 9 from a third column of liquid 13. The third column of liquid may be _ g _ ~~~~Qa4 PATENT
isotonic salt solution or cryoprotective solution.
Typically, the third or top column 13 contains the isotonic salt solution. The overall ratio of the isotonic salt solution to the cryoprotective solution in the container is in the range of about 3:1 to 4:1. The upper end of the tubular container is heat sealed by melting the end of the plastic straw to form a seal 15.
The solutions in the third column 13 and first column 6 of the container help to assure that the embryo is expelled from the gun when transferred to the recipient.
One preferred technique for loading the container shown in FIG.1 is to use a syringe to first draw the isotonic salt solution into the container to form the first column 6, then to allow the container to aspirate air bubble 8. A syringe is used to introduce the volume 9 of cryoprotective agent containing the embryo 10 into the container. Next, the container should be allowed to aspirate another air bubble 12, and finally the isotonic salt solution or the cryoprotective solution 13 should be drawn into the container with the syringe. Continued aspiration with the syringe will cause the isotonic salt solution in column 6 to contact layers 5 and 4 so that the sealant 4 solidifies. The container can then be heat sealed.
When the cryoprotective container is ready to be loaded into the artificial insemination gun, the heat sealed end 15 can be cut off and then the tubular container can be placed into the artificial insemination gun. The artificial insemination gun pushes the sealing plug 4 through the interior diameter of the tubular container forcing all of the liquid material in the container out its open end directly into the recipient animal.
Freezing the embryos involves first cooling them in a controlled rate freezer. The embryos can be either placed directly into the freezing apparatus at a temper-ature slightly below the freezing point of the PATENT
cryoprotective solution, which is called the seeding temperature, or can be cooled at a controlled rate of about -1°C/minute to the seeding temperature. The seeding temperature is normally in the range of about -5°C to -8°C. The embryos are held at this seeding temperature long enough to reach thermal equilibrium between the container and the freezer chamber which usually takes from about 1 to about 2 minutes. Ice nucleation is then induced by touching the container with a metal instrument previously supercooled in liquid nitrogen. The embryos are then cooled further at a controlled rate in the range of about -0.1°C/minute to about -1.0°C/minute until the embryos are in the range of about -2 0 ° C to -40 ° C . One pref erred method of the invention is to further cool the embryos after ice nucleation at the controlled rate of -0.3°C/minute to -0.5°C/minute to an end point temperature in the range of about -25°C to about -35°C. Once the embryos reach the end point temperature, they can be plunged immediately into liquid nitrogen or held at the endpoint temperature for a varying period of time prior to plunging, the preferred length of the hold being 15 minutes.
When the time comes to remove the embryos from storage in liquid nitrogen for transfer to recipient animals, the thawing procedure chosen should be suitable for the freezing procedures employed; i.e., the insulat-ing properties of the container, the cooling rates used and the terminal temperature at which the embryo is plunged into liquid nitrogen. It_is generally accepted that when an embryo is slow cooled to an endpoint temperature below -40°C (usually to -60°C to -80°C) before plunging the embryo into liquid nitrogen, a slow thaw rate (relative to the thaw rate required if the embryo is plunged into liquid nitrogen at a temperature greater than -40°C) is required to maintain embryo viability.
The thawing procedures for embryos plunged in liquid nitrogen at temperatures greater than -40°C may range from placing the cryoprotective container in air at room temperature to placing the container in a water bath of up to 40°C. In the preferred method of this invention the cryoprotective containers are placed into a water bath of about 30°C for about seconds.
As discussed before, the thawed embryo is then directly transferred to the recipient animal. In the preferred 10 method, this transfer is done immediately upon removal of the container from the water bath. Generally, this is done by loading the cryoprotective container into an artificial insemination gun and using the gun to deposit the embryo into the uterus of a suitable recipient female. Furthermore, the direct transfer step may include transferring the embryo from the cryoprotective container to an in vitro culture system and then, at a desired stage in its development, transferring the embryo to a recipient animal. The ultimate recipient is an animal of the same species as the embryo. An intermediate gestation may involve a suitable surrogate of another species.
In a preferred embodiment the invention provides a method for embryonic preservation of a bovine embryo or an embryo of another mammalian species, comprising a method for embryonic preservation for subsequent culture comprising the steps of: collecting Day 7 to 7 1/2 embryos in the late morula to expanded blastocyst stage; placing the embryos in a cryoprotective solution of from about 1.0 M to about 2.0 M
ethylene glycol and PB1 in plastic semen straws; allowing the embryos to equilibrate for about 10 minutes at room temperature; cooling the embryos in an alcohol bath freezer at -7°C for 2 minutes; inducing ice nucleation by touching the straw with a supercooled metal instrument; holding the embryos for 5 minutes at -7°C; cooling at a controlled rate of -0.5°C/minute to a final temperature of -35°C; holding at -35°C
for 15 minutes; plunging the embryos into liquid nitrogen for long term storage; thawing the embryos by placing the straws into a water bath of about 30°C for 10 seconds; and transferring the thawed embryos directly to a recipient mammalian female using an artificial insemination gun suitable for embryo transfer.
The following examples are provided to facilitate the understanding of preferred methods of the present invention and not for the purpose of limiting same. Those skilled in the art will recognize that various modifications in the procedure previously discussed and outlined below can be used for the purpose of practicing the present invention.
Bovine embryos can be collected in the range of Day 6 to Day 8 of the donor estrous cycle in the morula to hatched blastocyst stage, but in the preferred method are collected at Day 7 to Day 7 1/2 in the late morula to expanded blastocyst stage, placed in a cryoprotective solution of 1.5 M ethylene glycol in PB1, and loaded - 12a -PATENT
into a 0.25 ml plastic semen straw with a section of a 0.5 ml semen straw fitted over the sealed end for use as a handle and a labelling surface (Continental Plastics or I.M.V.). Next the embryos are allowed to equilibrate for ten minutes at room temperature. The freezing procedure for the embryos involves cooling the embryos in a controlled rate freezer to -7°C for two minutes, inducing ice nucleation by touching the straw with a supercooled metal instrument such as metal forceps, holding the embryos for 5 minutes at -7°C, cooling the embryos at a controlled rate of -0.5°C/minute to an end point temperature of -35°C, holding the embryos at -35°C
for 15 minutes, and then plunging the embryos into liquid nitrogen for final storage.
At the appropriate time for transfer of the embryos to the recipient animal, the embryos are thawed by placing the semen straws into a water bath of about 30°C
for ten seconds. The top end of the straw is then snipped off just below the heat seal and the straw is inserted into an artificial insemination gun. The entire contents of the straw are transferred to the uterus of an appropriate recipient animal by causing the plunger of the artificial insemination gun to push the plug at the bottom end of the straw through the length of the straw delivering the liquid contents and embryo out the open cut off end of the straw into the uterus of the animal.
Tables 1 and 2 show the results of this preferred method of the invention. The two tables complement each other in that Table 1 shows viability rates of the embryos after direct transfer to an in vitro culture, while Table 2 shows pregnancy rates resulting from direct transfer to a recipient cow. In these tables and the subsequent tables, the resul..ts of the preferred method are compared to a control of 10~ glycerol using a three-step rehydration procedure unless otherwise specified. This control was chosen because of the PATENT
widespread use of glycerol and step-wise rehydration methods in the cryoprotective field.
Table 1 below shows bovine embryo viability at 24, 48 and 72 hours for embryos in a culture medium with the number of viable embryos compared to the number of embryos frozen and the percentage of viable embryos listed in parentheses. Embryos frozen in glycerol were rehydrated using a step-wise procedure. Embryos frozen in 1.5 t4 ethylene glycol were transferred directly into tissue culture media (TCM) 199 with 10$ FCS.
TABLE 1. IN VITRO DEVELOPMENT OF EMBRYOS FROZEN
IN ETHYLENE GLYCOL AND REHYDRATED DIRECTLY
No. viable/No, frozen ($) Cryoprotectant 24 hours 48 hours 72 hours 10~ Glycerol 6/7 (86) 6/7 (86) 6/7 (86) 1.5 M Eth. Gly. 24/25 (96) 23/25 (96) 22/25 (88) These results demonstrate that embryos have a high survival rate with direct rehydration when frozen in 1.5 M ethylene glycol.
Table 2 below shows the number of bovine pregnancies compared to the number of embryos transferred to recipients for straws loaded with different solutions. The first column of data is for a straw loaded with 1.5 M ethylene glycol, in all three columns, (columns 6, 9 and 13 of Figure 1), while the second column of data shows pregnancy rates for a straw loaded with 1.5 M ethylene glycol in columns 9 and 13 of Figure 1 and PB1 in column 6 with the ratio of ethylene glycol to PB1 being 1:3. The third column of data is PATENT
for a straw loaded with 10~ glycerol in all three columns. Embryos frozen in glycerol were rehydrated using a three-step procedure. All others were transferred directly following thawing.
TABLE 2. PREGNANCY RATES AT ABOUT
No. pregnant/No. transferred within the indicated straw solutions Replicate 1.5 M Eth.Gly. 1.5 M Eth.Gly/PB1 10~ Glycerol Total 10/26 12/24 20/35 (38$) (50~) (57~) The results in Table 1 indicate higher viability for embryos in 1.5 M ethylene glycol rather than 10$
glycerol after direct transfer to an in vitro culture.
The data on pregnancy rates in Table 2 shows similar results for 1.5 M ethylene glycol and 10$ glycerol, especially when the straw is loaded with ethylene glycol and the PB1 in a 1:3 ratio.
Of the 22 pregnancies resulting from the direct transfers shown in Table 2, two have been allowed to proceed as normal pregnancies, and live calves are expected on about June 24, 1990. The other 20 preg-nancies were proceeding normally at greater than 40 days of gestation and were purposely aborted in order to reuse the recipient females in subsequent trials.
PATENT
The following is an alternative method using a cryopreservation pretreatment of trypsin.
This method is accomplished by following the steps of Example 1 with the additional step of exposing the embryos to a solution containing trypsin in the range of about 0.05 to 0.5~ in an isotonic salt solution prior to equilibrating the embryos in the cryoprotective solution. The embryos are washed in trypsin using a procedure similar to the one recommended by the Interna-tional Embryo Transfer Society (IETS). This procedure involves washing the embryos five times in Dulbecco's phosphate buffered saline with 0.4~ bovine serum albumin (BSA), washing the embryos two times (60 to 90 seconds each) in 0.25 trypsin in calcium and magnesium-free Hank's balanced salt solution (HBSS), and then washing the embryos five times in calcium and magnesium free Dulbecco's phosphate buffered saline with 10$ fetal calf serum (FCS).
Table 3 shows the effect of trypsin washing on the in vitro development of embryos frozen in ethylene glycol and rehydrated directly in a culture medium. In each column of data the number of viable bovine embryos at 24, 48 and 72 hours is compared to the number thawed and rehydrated directly. The percentage of viable embryos is shown in parentheses.
?(13~0~4 PATENT
TABLE 3. EFFECT OF TRYPSIN WASHING ON IN VITRO
DEVELOPMENT OF EMBRYOS FROZEN
IN ETHYLENE GLYCOL AND REHYDRATED DIRECTLY
No. viable/No. thawed (~) Treatment 24 hours 48 hours 72 hours Trypsin washed 1.5 M ethylene glycol Rep I 14/19 (74) 14/19 (74) 12/19 (63) Rep II 16/21 (76) 16/21 (76) 16/21 (76) Total 30/40 (75) 30/40 (75) 28/40 (70) Control washed 1.5 M ethylene glycol Rep I 13/19 (68) 13/19 (68) 8/19 (42) Rep II 19/21 (90) 19/21 (90) 18/21 (86) Total 32/40 (80) 32/40 (80) 26/40 (65) Embryos in replicate I were generally of poor quality which may explain their poor development compared with other ethylene glycol experiments.
Replicate II embryos were 7.5 to 8.0 day blastocysts.
Looking at replicate II, better results are achieved without a trypsin wash, but the viability rates are good for both trypsin washed and control washed embryos. The total figures indicate better results when the embryos are trypsin washed.
The following example is one preferred embodiment of the present invention in which the steps of Example 1 are followed and the direct transfer step includes the additional step of transferring the embryo from the cryoprotective container to an in vitro culture system -~ 2~3~~~4 PATENT
of PBl for a desired time period prior to transferring to the recipient animal. After transfer from the straw to PB1 the embryo can be briefly observed to assess viability and then loaded into another container for transfer to a recipient or held in culture for an extended period of time prior to transfer. In the case of the latter, the culture system would preferably consist of a bicarbonate buffered medium generally accepted for use in long-term culture of embryos and with or without feeder cells. Another option would be to transfer the embryos to the oviduct of an intermedi-ate host for temporary culture prior to transfer to a final recipient.
The following example illustrates varying concen-trations of the preferred cryoprotectant, ethylene glycol. The steps of Example 1 were followed. The cryoprotectant is ethylene glycol in the range of about 1.0 M to about 2.0 M in PB1.
Table 4 below shows bovine embryo viability at 24, 48 and 72 hours for different concentrations of ethylene glycol in the cryoprotective solution.
~~~~a~
PATENT
TABLE 4. IN VITRO DEVELOPMENT OF EMBRYOS FROZEN IN
1.0 M, 1.5 M, AND 2.0 M ETHYLENE GLYCOL
AND REHYDRATED DIRECTLY
No. viable/No. frozen Cryoprotectant 24 hours 48 hours 72 hours 10~ Glycerol 5/8 (63) 5/8 (63) 5/8 (63) 1.0 M Eth.Gly. 14/20 (70) 12/20 (60) 11/20 (55) 1.5 M Eth.Gly. 17/20 (85) 17/20 (85) 16/20 (80) 2.0 M Eth.Gly. 8/25 (32) 12/25 (48) 8/25 (32) The preferred concentration is about 1.5 M ethylene glycol based on this example's data. Embryos frozen in the glycerol control were rehydrated using a step-wise procedure.
Another alternative method of the present invention is shown in the following example. The steps of Example 1 are followed using different cryoprotective solutions of 10~ glycerol, 1.5 M propylene glycol in PB1 and 1.5 M
DMSO in PB1.
Table 5 below shows bovine embryo viability at 24, 48 and 72 hours for these cryoprotective solutions.
TABLE 5. IN VITRO DEVELOPMENT OF
- EMBRYOS FROZELJ IN GLYCEROL, ETHYLEPJE GLYCOL, PROPYLENE GLYCOL OR DMSO AND REHYDRATED DIRECTLY
No. (~) viable Cryoprotectant 24 hours 48 hours 72 hours 10'~ Glycerol 6/10 (60) 3/10 (30) 3/10 (30) 1.5 M Eth.Gly. 16/20 (80) 15/20 (75) 14/20 (70) 1.5 M DMSO 7/20 (35) 7/20 (35) 5/20 (35) 1.5 M Prop.Gly. 3/19 (16) 3/19 (i6) 2/19 (11) All embryos were placed directly into holding media after thawing. This experiment showed that the direct rehydration procedure does work with other cryoprotectants but not as well as with ethylene glycol.
The embryos used for the direct transfer technique can be developed using nuclear transfer techniques. The donor cell, typically an embryonic cell, is fused to an enucleated egg thereby transferring the nuclear material. A nuclear transfer procedure for bovine embryos is described in Bovine Nuclear Transplantation, Massey and Willadsen, PCT published application WO
88/09816, 15 Dec. 1988. -Those skilled in the art are familiar with other nuclear transfer procedures.
Table 6 shows in vitro development of cloned embryos produced by nuclear transfer frozen in 10~
glycerol and rehydrated using the step-wise procedure compared to cloned embryos produced by nuclear transfer, ~o~~o~~
PATENT
frozen in 1.5 M ethylene glycol, thawed, and rehydrated directly in the culture medium. Embryo viability is shown at 24, 48 and 72 hours.
TABLE 6. IN VITRO DEVELOPMENT OF EMBRYOS
PRODUCED BY NUCLEAR TRANSFER, FROZEN IN ETHYLENE GLYCOL, AND REHYDRATED DIRECTLY
No. ($) viable Cryoprotectant 24 hours 48 hours 72 hours 10$ Glycerol 14/27 (52) 13/27 (48) 13/27 (48) 1.5 M Eth.Gly. 18/28 (64) 14/28 (50) 14/28 (50) Embryos frozen in glycerol were rehydrated using a step-wise procedure. Embryos frozen in 1.5 M ethylene glycol were placed directly into a cell culture media of tissue culture media (TCM) 199 with 10$ FCS. The data shows that the direct transfer procedure is successful on nuclear transfer embryos.
Table 7 shows the pregnancies achieved in a first trial of direct transfer of cloned embryos produced by nuclear transfer to recipient females.
TABLE 7. PREGNANCY RATES OF DIRECT TRANSFER TRIALS
USING NUCLEAR TRANSFER EMBRYOS
Cryoprotectant- No. regnant/No transferred (~) 10~ Glycerol 1/11 (9) 1.5 M Ethylene Glycol 3/11 (27) Embryos frozen in glycerol were rehydrated using a step-wise procedure. All others were transferred directly following thawing. This data illustrates that the direct transfer procedure has value for cryopreser-vation of nuclear transfer embryos. The three recipient females pregnant with calves produced from direct transfer of nuclear transfer embryos are presently pregnant and will be maintained until parturition in about August and September of 1990. Therefore, a total of 5 pregnancies from the use of direct transfer proce-dures are presently being maintained.
The embryos used for the direct transfer technique can be developed using embryos that are cultured in vitro for part or all of the time to the desired age for cryopreservation. The fertilized eggs are placed in the appropriate culture medium for development. Many techniques and culture media are.known in the art. A
method for bovine embryo development is described in Bovine Embryo In Vitro Culture, Bondioli, PCT published Application WO 89/07135, 10 Aug. 1989.
References to other embryo culture systems are included in WO 89/07135 and are exemplary of alternative methods.
~--PATENT
The direct transfer method of this invention can be used with embryos that have been developed from an in vitro matured oocyte. The immature oocytes are collect-ed and cultured prior to fertilization. Various tech-niques have been known to those skilled in the art as described in Edwards, "Maturation In Vitro of Mouse, Sheep, Cow, Pig, Rhesus Monkey and Human Ovarian Oocytes," Nature, Vol. 208, pp. 349-351 (1965). More recently culture systems have been developed for various mammalian species including bovine oocytes. See e-g., Lu et al., "Pregnancy Established in Cattle by Transfer of Embryos Derived from In Vitro Fertilization of Oocytes Matured In Vitro," Vet. Rec., Vol. 121, pp.
259-260 (1987). Any successful in vitro oocyte matura-tion process can be used to produce the embryos to be frozen and subsequently transferred according to the method of this invention.
Those skilled in the art upon reading the above detailed description of the present invention will appreciate that many modifications of the method de-scribed above can be made without departing from the spirit of the invention. All such modifications which fall within the scope of the appended claims are intend-ed to be covered thereby.
Claims (21)
1. A method for embryonic preservation for subsequent culture comprising the steps of (a) collecting embryos;
(b) placing the embryos in a cryoprotective container with a cryoprotectant from a group consisting of ethylene glycol, dimethyl sulfoxide, glycerol, and any combinations thereof in a cell culture media of an isotonic salt solution;
(c) allowing the embryos to equilibrate in the cryoprotective solution;
(d) freezing the embryos for a desired time period;
(e) thawing the embryos; and (f) directly transferring the thawed embryos.
(b) placing the embryos in a cryoprotective container with a cryoprotectant from a group consisting of ethylene glycol, dimethyl sulfoxide, glycerol, and any combinations thereof in a cell culture media of an isotonic salt solution;
(c) allowing the embryos to equilibrate in the cryoprotective solution;
(d) freezing the embryos for a desired time period;
(e) thawing the embryos; and (f) directly transferring the thawed embryos.
2. A method of claim 1 wherein the embryos collected in step (a) are in the morula to hatched blastocyst stage.
3. A method of claim 1 wherein the embryos collected in step (a) are from artificially inseminated animals.
4. A method of claim 1 wherein the embryos collected in step (a) are produced by fertilization of in vitro matured oocytes.
5. A method of claim 1 wherein the embryos collected in step (a) are produced by in vitro fertilization of oocytes.
6. A method of claim 1 wherein the embryos collected in step (a) are produced by in vitro culture of fertilized oocytes.
7. A method of claim 1 wherein the embryos collected in step (a) are produced by nuclear transfer.
8. A method of claim 1 wherein the embryos collected in step (a) are produced by embryo splitting.
9. A method of claim 1 wherein the isotonic salt solution in step (b) has an osmolality of 250 to 350 milliosmoles, a pH in the range of 6.8 to 7.6, a pH buffer, and is supplemented from a group consisting of protein sources, synthetic macromolecules, carbohydrates, amino acids and vitamins.
10. A method of claim 1 wherein the embryos in step (b) are placed with the cryoprotectant in containers suitable for cryopreservation that provide for direct transfer to recipient animals.
11. A method of claim 1 including the additional step of exposing the embryos to a solution containing about 0.05% to 0.5% trypsin in an isotonic salt solution prior to step (c), equilibration in the cryoprotective solution.
12. A method of claim 1 including the additional step of exposing the embryos to a pretreatment of other agents selected from the group consisting of proteolytic enzymes, detergents, and organic solvents.
13. A method of claim 1 including the additional step of combining the embryos with pieces of trophoblastic tissue from a different viable embryo prior to step (c).
14. A method of claim 1 wherein the embryos are allowed to equilibrate in step (c) f or about 5 to about 40 minutes temperature in the range of from above 0°C to 39°C.
15. A method of claim 1 wherein the freezing step (d) includes:
cooling the embryos in a controlled rate freezer by a procedure selected from the group consisting of (a) placing embryos directly into the freezing apparatus at the temperature slightly below the freezing point of the cryoprotective solution which is called the seeding temperature and (b) cooling tile embryos at a controlled rate of about -1°C/minute to the seeding temperature;
holding the embryos at said temperature long enough to equilibrate the embryos at the seeding temperature; inducing ice nucleation by touching the container with a supercooled metal instrument;
continuing controlled rate cooling at about -0.1°C/min. to -1.0°C/min. to cool embryos to from about -20°C to about -40°C; and plunging the embryos into liquid nitrogen.
cooling the embryos in a controlled rate freezer by a procedure selected from the group consisting of (a) placing embryos directly into the freezing apparatus at the temperature slightly below the freezing point of the cryoprotective solution which is called the seeding temperature and (b) cooling tile embryos at a controlled rate of about -1°C/minute to the seeding temperature;
holding the embryos at said temperature long enough to equilibrate the embryos at the seeding temperature; inducing ice nucleation by touching the container with a supercooled metal instrument;
continuing controlled rate cooling at about -0.1°C/min. to -1.0°C/min. to cool embryos to from about -20°C to about -40°C; and plunging the embryos into liquid nitrogen.
16. A method of claim 1 wherein in step (e) the embryos in the cryoprotective solution are thawed in a water bath at a temperature of about 30°C.
17. A method of claim 1 wherein the direct transfer step (f) comprises transferring the embryo in the cryopreservation container directly to a recipient animal.
18. A method of claim 1 wherein the direct transfer step (f) comprises transferring the embryo from the cryoprotective container directly to an in vitro culture system and subsequently transferring the embryo to a recipient animal.
19. A method for bovine embryonic preservation for subsequent culture comprising the steps of:
collecting Day 7 to 7 1/2 bovine embryos in the late morula to expanded blastocyst stage;
placing the embryos in a cryoprotective solution of from about 1.0 M to about 2.0 M
ethylene glycol and PB1 in plastic semen straws;
allowing the embryos to equilibrate for about 10 minutes at room temperature;
cooling the embryos in an alcohol bath freezer at -7°C for 2 minutes;
inducing ice nucleation by touching the straw with a supercooled metal instrument;
holding the embryos for 5 minutes at -7°C;
cooling at a controlled rate of -0.5°C/minute to a final temperature of -35°C; holding at -35°C
for 15 minutes;
plunging the embryos into liquid nitrogen for long term storage;
thawing the embryos by placing the straws into a water bath of about 30°C for 10 seconds; and transferring the thawed embryos directly to a recipient cow using an artificial insemination gun suitable for embryo transfer.
collecting Day 7 to 7 1/2 bovine embryos in the late morula to expanded blastocyst stage;
placing the embryos in a cryoprotective solution of from about 1.0 M to about 2.0 M
ethylene glycol and PB1 in plastic semen straws;
allowing the embryos to equilibrate for about 10 minutes at room temperature;
cooling the embryos in an alcohol bath freezer at -7°C for 2 minutes;
inducing ice nucleation by touching the straw with a supercooled metal instrument;
holding the embryos for 5 minutes at -7°C;
cooling at a controlled rate of -0.5°C/minute to a final temperature of -35°C; holding at -35°C
for 15 minutes;
plunging the embryos into liquid nitrogen for long term storage;
thawing the embryos by placing the straws into a water bath of about 30°C for 10 seconds; and transferring the thawed embryos directly to a recipient cow using an artificial insemination gun suitable for embryo transfer.
20. A method of claim 19 wherein the cryoprotective solution is comprised of 1.5 M ethylene glycol and PB1.
21. A method for embryonic cryopreservation and subsequent culture comprising the same steps as claim 19, except that embryos of other mammalian species will be used.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US47821690A | 1990-02-09 | 1990-02-09 | |
| US478,216 | 1990-02-09 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2034054A1 CA2034054A1 (en) | 1991-08-10 |
| CA2034054C true CA2034054C (en) | 2001-03-27 |
Family
ID=23899000
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2034054 Expired - Fee Related CA2034054C (en) | 1990-02-09 | 1991-01-11 | Cryopreservation process for direct transfer of embryos |
Country Status (4)
| Country | Link |
|---|---|
| CA (1) | CA2034054C (en) |
| IE (1) | IE910417A1 (en) |
| IL (1) | IL96906A0 (en) |
| NZ (1) | NZ236757A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR102016003292B1 (en) * | 2015-07-14 | 2022-08-09 | Genus Plc | METHOD TO PREPARE IN VITRO PRODUCED UNGULATE EMBRYO, DEVICE AND METHODS OF FREEZING AND THawING UNGULATE EMBRYOS |
-
1991
- 1991-01-08 IL IL96906A patent/IL96906A0/en unknown
- 1991-01-11 CA CA 2034054 patent/CA2034054C/en not_active Expired - Fee Related
- 1991-01-14 NZ NZ23675791A patent/NZ236757A/en unknown
- 1991-02-08 IE IE41791A patent/IE910417A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| IE910417A1 (en) | 1991-08-14 |
| CA2034054A1 (en) | 1991-08-10 |
| NZ236757A (en) | 1993-05-26 |
| IL96906A0 (en) | 1992-03-29 |
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