Note: Descriptions are shown in the official language in which they were submitted.
.-- 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
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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
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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
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..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
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..~ ~.~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
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.....
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
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~~~~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
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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.
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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
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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.
EXAMPLE 1
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
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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
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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 ($)
Cryoprotectant 24 hours 48 hours 72 hours
10~ Glycerol 6/7 (86) 6/7 (86) 6/7 (86)
1.5 M Eth. Gly. 24/25 (96) 23/25 (96) 22/25 (88)
These results demonstrate that embryos have a high
survival rate with direct rehydration when frozen in 1.5
M ethylene glycol.
Table 2 below shows the number of bovine
pregnancies compared to the number of embryos
transferred to recipients for straws loaded with
different solutions. The first column of data is for a
straw loaded with 1.5 M ethylene glycol, in all three
columns, (columns 6, 9 and 13 of Figure 1), while the
second column of data shows pregnancy rates for a straw
loaded with 1.5 M ethylene glycol in columns 9 and 13 of
Figure 1 and PB1 in column 6 with the ratio of ethylene
glycol to PB1 being 1:3. The third column of data is
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PATENT
for a straw loaded with 10~ glycerol in all three
columns. Embryos frozen in glycerol were rehydrated
using a three-step procedure. All others were
transferred directly following thawing.
TABLE 2. PREGNANCY RATES AT ABOUT
40 DAYS FOR DIRECT TRANSFER
No. pregnant/No. transferred within the
indicated straw solutions
Replicate 1.5 M Eth.Gly. 1.5 M Eth.Gly/PB1 10~ Glycerol
I 6/11 -- 8/11
II -- 5/9 4/9
III 4/15 7/15 8/15
Total 10/26 12/24 20/35
(38$) (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.
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PATENT
EXAMPLE 2
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.
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?(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.
EXAMPLE 3
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
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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.
EXAMPLE 4
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.
EXAMPLE 5
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.
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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.
EXAMPLE 6
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,
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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.
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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.
EXAMPLE 7
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.
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PATENT
EXAMPLE 8
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.
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