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The effect of cooling rate, warming rate, cryoprotective agent and stage of development of survival of mouse embryos during freezing and thawing
life Sdmces Voll l, Pert Q PP . 1071-1079 1972 Prlnbed in C~eat Britain
P~ergamon Press
THE EFFECT OF COOLING RATE, üARMING RATE, CRYOPROTECTIVE AGENT AND STAGE OF DEVELOPMENT ON SURVIVAL OF MOUSE EMBRYOS DURING FREEZING AND THARING I. Wilmut A.R .C . Unit of Reproductive Physiology and Biochemistry, 307 Huntingdon Road, Cambridge, England.
(Received 20 September 192 ; in final form 9 October 192) INTRODUCTION Ia the last txenty years many different techniques have been developed to achieve survival of cells and tissues during freezing and thawing.
Several
attempts have bases made to preserve maamalian eggs and embryos at very lox temperatures (1), but success has been very limited (2) .
Recently, however, it
has been reported that mouse blastocyats survived freezing and thawing in a medium which contained polyvinylpyrrolidone (PVP) as a protective agent (3) . Experiments at these laboratories completely failed to substantiate this claim sad further studies have therefore been carried out to examine factors affecting the survival of mouse embryos at low temperatures . ühen cells are frozen and thawed the factors which are most important in determining their survival are the type of cell, rate of cooling, rate of xarming and the composition of the medium . obese factors (4) .
There are also interactions between
Compounds which have been shown to have cryoprotective
activity range from inorganic salts (5), to sugars and alcohols (6,7,8), amides (6) and some polymers such as PVP and dextran (9,10) .
They can usefully be
grouped into three categories according to their ability to enter cells and their osmotic effects .
One compound from each category was chosen for invea
tig8tioa is these experiments .
Dimethylsulphoxide (DMSO) was chosen as a com-
pound able to penetrate readily into cells, sucrose as a compound not able to penetrate into cells, but which exerts a significant osmotic effect and finally PVP as a non-permeating polymer which has an insignificant dehydrating effect at the concentrations employed .
These compounds
1071
Survival of Fmbryo afba Freezing
1072
Vo1.l1,No. 22
were examined at different concentrations for their toxicity to mouse embryos and for their effectiveness as cryoprotective agents during cooling and warming at various rates . MATERIALS AND METHODS Embryos :
Female albino mice of the non-inbred Carworth Europe strain CFLP,
aged 5 to 9 weeks, were induced to superovulate by intraperitoneal injection of 5 to 10 iu pregnant mares'
serum followed 46 to 50 hr later by 5 to 10 iu
human chorionic goaadotrophin (HCG) .
Two or three females were caged with a
male at the time of the HCG injection and those having vaginal plugs the following morning were used as donors .
Eight-cell eggs and early blastocysts were
recovered 68 to 72 hr and 91 to 96 hr after the injection of HCG, respectively . Dulbecco's phosphate-buffered salt solution, enriched with energy-sources and bovine serum albumin (3) was used to flush the reproductive tracts immediately after autopsy .
The concentration of albumin was 4 mg/ml and 50 ug/ml strepto-
mycin sulphate was also included . Freezing and thawing:
Embryos were frozen and thawed in 50 x 6 mm glass
test tubes containing 0 .2 ml of phosphate-buffered medium .
The protective
agents were incorporated in phosphate-buffered media and the pH of such media was adjusted to 7 .2 .
The principle of a method by which different cooling
rates can be obtained has been described by others (11,12) and the apparatus used in these experiments and the different cooling and warming rates which were achieved are described in Tables 1 and 2 .
The tubes were transferred
from room temperature to crushed ice and after 15 min to a seeding bath which was cooled to a temperature approximately 2° C below the freezing point of the medium .
Ice formation was induced by seeding with an ice crystal and 5 min
later the samples were placed in the cooling vessels .
These vessels were
cooled in liquid nitrogen and at the time of the transfer had cooled to the temperature of the seeding bath .
When the temperature of the samples reached
-70oC they were transferred to liquid nitrogen where they were stored for 12 hr to 12 days .
Temperature changes were measured with a Cambridge DE Electronic
9mvival of Fmbryo after Freezing
Vo1.11,No. 22
1073
Recorder fitted with a copper-constantan thermocouple, and the ratés given are those measured between -10°C and -70°C. TABLE 1 The cooling rates which were obtained when the samples were transferred to various containers as they were cooled in liquid nitrogen* Cooling rate (-10 to -70°C)
Container
0.07°C/min
An evacuated silvered flask, internal measurements 100 x 200 mm containing 850 ml ethanol .
0.22°C/min
An evacuated unsilvered flask, internal measurements 100 x 200 mm containing 400 ml ethanol .
0.67°C/min
400 ml double walled beaker, internal measurements 80 x 100 mm containing 150 ml ethanol.
1.2 °C/min
Empty evacuated unailvered flask, internal measurements 100 x 200 mm .
4 .7 °C/min
400 ml double walled plastic beaker containing 150 ml ethanol.
23° C/min
Empty 250 ml pyrex beaker .
80°C/min
25 x 150 mm pyrex tube pre-cooled in liquid nitrogen .
690°C/min
Tubes placed directly in liquid nitrogen . TABLE 2
The warming rates which were obtained when the samples were taken from liquid nitrogen and placed in the containers* Flarming rate (-70 to -10°C)
Container
1.1°C/min
1 litre pyrex beaker containing 600 ml ethanol cooled to -70° C. The beaker was allowed to warm in air .
12°C/min
16 x 125 mm pyrex tube which had been cooled in liquid nitrogen . The tube was allowed to warm in air .
60°C/min
Tubes shaken in air at room temperature .
360°C/min
Tubes shaken in water at 37°C .
* The contents of all the containers except those used to achieve cooling rates 80 or 690°C/min or warming rates of 12, 60 or 360°C/min, were stirred . Assessment of survival :
The embryos xere cultured by the microdrop method
in a modified Krebs-Ringer bicarbonate medium at 37 °C under a gas-phase of 5$ C02 in air,
(13) .
Eight-cell eggs were cultured for 48 hr and early blasto-
cysts for 24 hr, and in both cases the criterion used to assess survival was whether or not the embryo was able to develop to a fully expanded blastocyst .
VoL 11,No. 22
Survival of Frnbryo after Freezing
1074
The results presented are the number of embryos developing expressed as a percentage of the number cultured .
A proportion of the embryos was lost from
each treatment during freezing and thawing and it has been assumed that those lost were a random sample of the population being frozen and thawed . RESULTS Effects of the agents at 20°C :
PVP is a polymer with a considerable
range of molecular weights, and even pharmaceutical grade preparations have been shown to contain appreciable quantities of low molecular weight contamin ants (14) .
As dialysis removes a factor in PVP which is toxic to Pseudomonas
F8 during freezing and thawing (14), a comparison was made of the effects of dialysed and non-dialysed preparations .
Twenty grammes of PVP (Sigma, Pharma-
ceutical grade MW 40,000) were dissolved in 200 ml distilled water and dialysed at 4°C for 20 hr against three volumes of distilled water each of 15 litres . The dialysed material was then freeze dried .
Early blastocysts were incub-
ated in the presence of 7 .5$ (w/v) PAP at 20°C for 4 hr, before being recovered, rinsed thoroughly and cultured .
They were compared with controls
which had been incubated for 4 hr at 20°C in the phosphate buffered medium . Other embryos were placed directly into medium containing 0 .6 M-sucrose and incubated in this medium for 30 min before being recovered, rinsed thoroughly and cultured .
4lhen medium containing DMSO was added to embryos at 20°C they
shrank and then re-expanded in approximately 5 min .
Preliminary experiments
showed that direct addition of medium containing 2 .0 M-DMSO irreversibly damaged the embryos, and that prolonged exposure to DMSO was also damaging . The optimal rate of addition appeared to be in increments of 0 .5 M to 1.0 M at 5 to 10 min intervals .
A group of embryos was exposed to 1 .5 M-DMSO which was
added in three steps of 0.5 M at 10 min intervals and after 30 min was removed in three steps before the embryos were rinsed and cultured . These concentrations of the protective agents were chosen because they have been shown to provide some protection for other cells (10,15) .
The
results are presented in Table 3 and it is apparent that development was not
VoL 11,No. 22
1075
Survival of Fmbryo after Freezing
affected by exposure to DMSO, sucrose or dialysed PVP, but that exposure to undialysed PVP reduced the proportion which developed (P<0 .001) . TABLE 3 The effect of 1.5 M-DMSO, 0.6 M-sucrose and 7 .5$ PVP on the subsequent development of early blastocysts . (See text for details of treatments) . Number of blastocysts
Number developing
Percentage developing
Control DMSO
80 80
77 75
96 .3 93 .8
Control Sucrose
60 60
49 51
81 .7 85 .0
Control Dialysed PVP Undialysed PVP
75 75 75
66 64 1
88 .0 85 .3 1 .3
Groups of 20 blastocysts were frozen and thawed at
EY~eezing and thaw~ng :
four different cooling rates in the presence of 7 .5$ dialysed PVP, 0 .6 M-sucrose or 2 .0 M-DMSO .
The thawing rate was 360° C/min .
After thawing PVP and
sucrose were removed in one step, the DMSO was removed in three steps and the embryos rinsed thoroughly before being cultured .
No blastocysts developed
after being frozen and thawed in the presence of PVP or sucrose, whereas 1 of 18 and 3 of 17 expanded after being cooled at 23 °C/min and O .67oC/min respectively, in the presence of 2.0 M-DMSO (Table 4) . TABLE 4 The effect of cooling rate and cryoprotective agent on the survival of frozen and thawed early blaetocysta . The percentage which developed is shown and, in parentheses, the number of blastocyata cultured . Protective agent
Cooling rate . °C/min 23 80
0 .67
690
Sucrose
0
(41)
0
(40)
0 (42)
0 (42)
PVP
0
(24)
0
(65)
0 (68)
0 (67)
5.6 (18)
0 (19)
0 (19)
DMSO
17 .6 (17)
The effect of warming rate was examined after cooling groups of 20 blastocysts at four different rates in the presence of 1 .5 M-DMSO .
An interaction
between cooling and warming rates was apparent (Table 5), with optimum warming
Vo1 .11,No . 22
Si~rvival of Flnbryo after Freezing
1076
rate becoming slower as the cooling rate was reduced . Maximum survival, which was 64 .7$, was obtained with a cooling rate of 0 .22°C/min and a warming rate of 12 °C/min .
After cooling at 0 .22° C/min and
warming at 1 .1 and 12 °C/min, 22 .5 and 64 .7$ of the blastocysts survived respactively .
The "exact probability" of such a large difference occurring by
chance in the absence of an effect of warming rate was found to be 0.033, which suggests that warming rate has a significant effect .
The exact test
was applied because of the small numbers of eggs in each treatment . TABLE 5 The effect of cooling rate and warming rate on the survival of early blastocysts frozen and thawed in the presence of 1.5 M-DMSO . The percentage which developed is shown, and, in parentheses, the number cultured . üarming rate °C/min 1.1
0.07
Cooling rate 0 .22
25 .0 (16)
12
0
(16)
3so
o
(ls)
°C/min 1 .2
4 .7
22 .5 (13)
0
(17)
0 (19)
64 .7 (17)
0
(19)
0 (18)
12 .s (ls)
o (ls)
o
(le)
Groups of 80 blastocysts were cooled at 0.22°C/min in the presence of 1.5 M-DMSO and warmed at 1 .1, 12 or 60°C/min.
The percentages which survived
following these three warming rates were 53 .8 (42 of 78), 85 .7 (66 of 77) and 64 .7 (44 of 68) respectively .
Comparisons of these waxing rates by Chi-
squared tests showed that 12 °C/min was the optimal rate and that 1 .1 and 60°C/ min were significantly different from each other.
(1 .1 v 12 °C/min P<0 .001 ;
12 v 60 °C/min P<0 .01) . Finally, when eight-cell eggs were cooled at 0.22°C/min and warmed at 12 °C/min, 65 .8$ developed (27 of 41) .
As 84 .0$ of untreated eggs developed
to blastocysts this represents a survival of 78 .4$ . DISCUSSION These experiments show clearly that a high proportion of mouse embryos, either at the eight-cell stage or as blastocysts, can survive freezing and storage in liquid nitrogen if they are suspended in medium containing DMSO .
Vo1 .11,No. 22
Survival of Fmbryo after Freezing
1077
So far viability has been demonstrated only by development in culture and it is important that this should be confirmed by transferring frozen and thawed embryos to recipient foster mothers .
Nevertheless, it seems probable that
these techniques will lead to the development of a practical method for the long term storage of embryos . The complete failure to obtain survival of blastocysts frozen and thawed in medium containing PVP in the present experiments confirms our previous observations and is again in contrast to the earlier report that survival was achieved when blastocysts xere held in this medium for 30 min in solid carbon dioxide (3) .
No valid explanation for this discrepâncy can be made .
Three observations of particular interest were made during these experimonts .
Firstly it xas noted that after freezing and thawing the cells in each
egg were usually either all damaged or all normal in appearance .
Furthermore,
similar proportions of eight-cell eggs and blastocysts survived freezing and thawing, which suggests that, in the main, freezing and thawing affected the embryos as a whole and not as individual cells xithin embryos .
Secondly, the
cooling rate which was found to be optimal was of the order expected either for very large cells or for cells which are, relatively, very impermeable to xater (17) .
Finally, it is very unusual for the optimum warming rate to be
as slow as 12 ° C/min .
In some circumstances, rapid warming of frozen plant
cells is found to be damaging (18), .and, in an isolated observation, elox warming of red blood cells was beneficial (19) .
Hoxever, with yeasts (11),
and hamster and mouse cells (12,15) rapid warming was never damaging and is some circumstances was clearly beneficial . Embryos at both of these stages of development are contained in a mucoprotein envelope, the zone pellucida, which has a diameter of approximately 100u, and it is possible that it is the presence of the zona which determines the optimum treatment.
Fihen the samples were seeded and ice formed, the
fluid outside the zona would have become hypertonic and equilibrium could only have been restored either by entry of solutes or by removal of water through
Survival of Fmbryo after Freezing
1078
the zone .
Vo1.11,No . 22
As the zones is relatively inflexible and unlikely to shrink, it
seems probable that movement of solutes restored equilibrium.
When the
cooling rate was sufficiently slow, ice would have been unlikely to form within the cells or the perivitelline space, as equilibrium could have been maintained by movement of solutes .
If such samples were warmed rapidly the
cells would have been exposed to a hypertonic environment at 20° C, whereas if the warming rate was slow, equilibrium could have been maintained by the loss of excess solutes from the perivitelline apace .
Thus, it is suggested that
because it determines the cooling rate at which ice formation in the perivitelline space or the cells becomes likely, it is the presence of the zone pellucida which determines that the optimum cooling and warming rates will be very slow . It has been established with a Chinese hamster cell that survival during freezing and thawing varies with the otage of the cell-cycle (20), and it is possible that the small number of dead cells which were observed in blastocysts which were fixed and stained after being frozen and thawed were those which were at a particularly sensitive phase of the cell-cycle when they were frozen and thawed . The practical value of techniques for long term storage of the embryos of large domestic animals has been stressed recently (21) .
Such techniques would
greatly facilitate the international exohange of genetically valuable livestock and could also provide a reservoir of embryos for use in the production of twins in cattle by a non-surgical transfer technique . I also I gratefully acknowledge receipt of a Milk Marketing Hoard Fellowship . wish to thank Dr . C. Polge for the provision of faciliti®s and for his advice, Dr . J. Ferrant for his advice during these experiments, Mr . D.E . Welters for advice on statistical analyses and Mr . K .T . Elsome for skilled technical assistance .
Vo1.11,No. 22
~
Sbrri~al of Fmbryo dta Freezing
1079
REFERENCES 1.
E.S .E . Hafez, Acta Endocrinol ., Suppl . 140, 24 (1969) .
2.
M. Ferdows, C .L . Moore and A.E . Dracy, J. Dairy Sci ., 41, 739 Abs . (1958) .
3.
D .G . Whittingham, Nature (Lund.), 233, 125 (1971) .
4.
P . Mazur, S .P . Leibo, J. Ferrant, E .H .Y . Chu, M .G . Hanna and L.H . Smith, p .69 in "The Frozen Cell" Ed . G.E .W . Wolstenholme and M . O'Connor : and A. Churchill ;
J.
London (970) .
5.
Phan The Tran and M.A . Bender, Proc . Soc . exp . Biol . Med., 104, 388 (1960) .
6.
J .E . Lovelock, Biochem. J ., 56, 265 (1954) .
7.
C . Polge, A.U . Smith and A.S . Parker, Nature (Lund .), 164, 666 (1949).
8.
C . Polge and M.A . Soltya p.87 in "Recent Research in Freezing and Drying" . Ed . A.S . Parker and A.U . Smith . Blackwells, Oxford (1959) .
9.
M. Persidaky and V. Richards, Nature (Land.), 196, 585 (1962),
10 .
G.F . Doebbler and A.P . Rinfret, Biochim. Biophys . Acte . 58, 449 (1962).
11 .
P . Mazur and J. Schmidt, Cryobiology , 5, 1 (1968) .
12 .
S.P . Leibo, J . Ferrant, P . Mazur, M.G . Hanna, and L .H . Smith, Cryobiology 6, 315 (1970) .
13 .
D.G . Whittingham, J. Reprod . Fert . Suppl . 14, 7 (1971),
14 .
M.J . Aahwood-Smith and C. Warby, Cryobiology , 8, 453 (1971) .
15 .
P. Mazur, J . Ferrant, S .P . Leibo and E .H .Y . Chu, Cryobiology , 6, 1 (1969) .
16 .
N .T .J . Bailey "Statistical Methods in Biology", English Universities Press, London (1959).
17 .
P . Mazur, Cryobiôlogy , 2, 181 (1966) .
18 .
J . Levitt, P . 495 in "Cryobiology" .
Ed . H .T . Meryman, Academic Press,
London (1966) . 19 .
H .T . Meryman P . 231 in "Cellular Injury and Resistance in Freezing Organisms" .
Ed . E. Ashanina and E . Sapporo .
ence, Hokkaido University (1967) . Cell" .
Institute of Low Temperature Sci-
Cited by J . Ferrant P . 295 in "The Frozen
Ed . G.E .W . Woletenholme and M. O'Connor .
J . and A. Churchill,
London (1970) . 20 .
G.J . Koch, J . Kruuv and C .W . Bruckschwaiger, Exp . Cell Res . 63, 476 (1970
21 .
L.E .A . Rnwson, J. Reprod . Fert . 26, 113 (1971) .
P~ergamon Press
THE EFFECT OF COOLING RATE, üARMING RATE, CRYOPROTECTIVE AGENT AND STAGE OF DEVELOPMENT ON SURVIVAL OF MOUSE EMBRYOS DURING FREEZING AND THARING I. Wilmut A.R .C . Unit of Reproductive Physiology and Biochemistry, 307 Huntingdon Road, Cambridge, England.
(Received 20 September 192 ; in final form 9 October 192) INTRODUCTION Ia the last txenty years many different techniques have been developed to achieve survival of cells and tissues during freezing and thawing.
Several
attempts have bases made to preserve maamalian eggs and embryos at very lox temperatures (1), but success has been very limited (2) .
Recently, however, it
has been reported that mouse blastocyats survived freezing and thawing in a medium which contained polyvinylpyrrolidone (PVP) as a protective agent (3) . Experiments at these laboratories completely failed to substantiate this claim sad further studies have therefore been carried out to examine factors affecting the survival of mouse embryos at low temperatures . ühen cells are frozen and thawed the factors which are most important in determining their survival are the type of cell, rate of cooling, rate of xarming and the composition of the medium . obese factors (4) .
There are also interactions between
Compounds which have been shown to have cryoprotective
activity range from inorganic salts (5), to sugars and alcohols (6,7,8), amides (6) and some polymers such as PVP and dextran (9,10) .
They can usefully be
grouped into three categories according to their ability to enter cells and their osmotic effects .
One compound from each category was chosen for invea
tig8tioa is these experiments .
Dimethylsulphoxide (DMSO) was chosen as a com-
pound able to penetrate readily into cells, sucrose as a compound not able to penetrate into cells, but which exerts a significant osmotic effect and finally PVP as a non-permeating polymer which has an insignificant dehydrating effect at the concentrations employed .
These compounds
1071
Survival of Fmbryo afba Freezing
1072
Vo1.l1,No. 22
were examined at different concentrations for their toxicity to mouse embryos and for their effectiveness as cryoprotective agents during cooling and warming at various rates . MATERIALS AND METHODS Embryos :
Female albino mice of the non-inbred Carworth Europe strain CFLP,
aged 5 to 9 weeks, were induced to superovulate by intraperitoneal injection of 5 to 10 iu pregnant mares'
serum followed 46 to 50 hr later by 5 to 10 iu
human chorionic goaadotrophin (HCG) .
Two or three females were caged with a
male at the time of the HCG injection and those having vaginal plugs the following morning were used as donors .
Eight-cell eggs and early blastocysts were
recovered 68 to 72 hr and 91 to 96 hr after the injection of HCG, respectively . Dulbecco's phosphate-buffered salt solution, enriched with energy-sources and bovine serum albumin (3) was used to flush the reproductive tracts immediately after autopsy .
The concentration of albumin was 4 mg/ml and 50 ug/ml strepto-
mycin sulphate was also included . Freezing and thawing:
Embryos were frozen and thawed in 50 x 6 mm glass
test tubes containing 0 .2 ml of phosphate-buffered medium .
The protective
agents were incorporated in phosphate-buffered media and the pH of such media was adjusted to 7 .2 .
The principle of a method by which different cooling
rates can be obtained has been described by others (11,12) and the apparatus used in these experiments and the different cooling and warming rates which were achieved are described in Tables 1 and 2 .
The tubes were transferred
from room temperature to crushed ice and after 15 min to a seeding bath which was cooled to a temperature approximately 2° C below the freezing point of the medium .
Ice formation was induced by seeding with an ice crystal and 5 min
later the samples were placed in the cooling vessels .
These vessels were
cooled in liquid nitrogen and at the time of the transfer had cooled to the temperature of the seeding bath .
When the temperature of the samples reached
-70oC they were transferred to liquid nitrogen where they were stored for 12 hr to 12 days .
Temperature changes were measured with a Cambridge DE Electronic
9mvival of Fmbryo after Freezing
Vo1.11,No. 22
1073
Recorder fitted with a copper-constantan thermocouple, and the ratés given are those measured between -10°C and -70°C. TABLE 1 The cooling rates which were obtained when the samples were transferred to various containers as they were cooled in liquid nitrogen* Cooling rate (-10 to -70°C)
Container
0.07°C/min
An evacuated silvered flask, internal measurements 100 x 200 mm containing 850 ml ethanol .
0.22°C/min
An evacuated unsilvered flask, internal measurements 100 x 200 mm containing 400 ml ethanol .
0.67°C/min
400 ml double walled beaker, internal measurements 80 x 100 mm containing 150 ml ethanol.
1.2 °C/min
Empty evacuated unailvered flask, internal measurements 100 x 200 mm .
4 .7 °C/min
400 ml double walled plastic beaker containing 150 ml ethanol.
23° C/min
Empty 250 ml pyrex beaker .
80°C/min
25 x 150 mm pyrex tube pre-cooled in liquid nitrogen .
690°C/min
Tubes placed directly in liquid nitrogen . TABLE 2
The warming rates which were obtained when the samples were taken from liquid nitrogen and placed in the containers* Flarming rate (-70 to -10°C)
Container
1.1°C/min
1 litre pyrex beaker containing 600 ml ethanol cooled to -70° C. The beaker was allowed to warm in air .
12°C/min
16 x 125 mm pyrex tube which had been cooled in liquid nitrogen . The tube was allowed to warm in air .
60°C/min
Tubes shaken in air at room temperature .
360°C/min
Tubes shaken in water at 37°C .
* The contents of all the containers except those used to achieve cooling rates 80 or 690°C/min or warming rates of 12, 60 or 360°C/min, were stirred . Assessment of survival :
The embryos xere cultured by the microdrop method
in a modified Krebs-Ringer bicarbonate medium at 37 °C under a gas-phase of 5$ C02 in air,
(13) .
Eight-cell eggs were cultured for 48 hr and early blasto-
cysts for 24 hr, and in both cases the criterion used to assess survival was whether or not the embryo was able to develop to a fully expanded blastocyst .
VoL 11,No. 22
Survival of Frnbryo after Freezing
1074
The results presented are the number of embryos developing expressed as a percentage of the number cultured .
A proportion of the embryos was lost from
each treatment during freezing and thawing and it has been assumed that those lost were a random sample of the population being frozen and thawed . RESULTS Effects of the agents at 20°C :
PVP is a polymer with a considerable
range of molecular weights, and even pharmaceutical grade preparations have been shown to contain appreciable quantities of low molecular weight contamin ants (14) .
As dialysis removes a factor in PVP which is toxic to Pseudomonas
F8 during freezing and thawing (14), a comparison was made of the effects of dialysed and non-dialysed preparations .
Twenty grammes of PVP (Sigma, Pharma-
ceutical grade MW 40,000) were dissolved in 200 ml distilled water and dialysed at 4°C for 20 hr against three volumes of distilled water each of 15 litres . The dialysed material was then freeze dried .
Early blastocysts were incub-
ated in the presence of 7 .5$ (w/v) PAP at 20°C for 4 hr, before being recovered, rinsed thoroughly and cultured .
They were compared with controls
which had been incubated for 4 hr at 20°C in the phosphate buffered medium . Other embryos were placed directly into medium containing 0 .6 M-sucrose and incubated in this medium for 30 min before being recovered, rinsed thoroughly and cultured .
4lhen medium containing DMSO was added to embryos at 20°C they
shrank and then re-expanded in approximately 5 min .
Preliminary experiments
showed that direct addition of medium containing 2 .0 M-DMSO irreversibly damaged the embryos, and that prolonged exposure to DMSO was also damaging . The optimal rate of addition appeared to be in increments of 0 .5 M to 1.0 M at 5 to 10 min intervals .
A group of embryos was exposed to 1 .5 M-DMSO which was
added in three steps of 0.5 M at 10 min intervals and after 30 min was removed in three steps before the embryos were rinsed and cultured . These concentrations of the protective agents were chosen because they have been shown to provide some protection for other cells (10,15) .
The
results are presented in Table 3 and it is apparent that development was not
VoL 11,No. 22
1075
Survival of Fmbryo after Freezing
affected by exposure to DMSO, sucrose or dialysed PVP, but that exposure to undialysed PVP reduced the proportion which developed (P<0 .001) . TABLE 3 The effect of 1.5 M-DMSO, 0.6 M-sucrose and 7 .5$ PVP on the subsequent development of early blastocysts . (See text for details of treatments) . Number of blastocysts
Number developing
Percentage developing
Control DMSO
80 80
77 75
96 .3 93 .8
Control Sucrose
60 60
49 51
81 .7 85 .0
Control Dialysed PVP Undialysed PVP
75 75 75
66 64 1
88 .0 85 .3 1 .3
Groups of 20 blastocysts were frozen and thawed at
EY~eezing and thaw~ng :
four different cooling rates in the presence of 7 .5$ dialysed PVP, 0 .6 M-sucrose or 2 .0 M-DMSO .
The thawing rate was 360° C/min .
After thawing PVP and
sucrose were removed in one step, the DMSO was removed in three steps and the embryos rinsed thoroughly before being cultured .
No blastocysts developed
after being frozen and thawed in the presence of PVP or sucrose, whereas 1 of 18 and 3 of 17 expanded after being cooled at 23 °C/min and O .67oC/min respectively, in the presence of 2.0 M-DMSO (Table 4) . TABLE 4 The effect of cooling rate and cryoprotective agent on the survival of frozen and thawed early blaetocysta . The percentage which developed is shown and, in parentheses, the number of blastocyata cultured . Protective agent
Cooling rate . °C/min 23 80
0 .67
690
Sucrose
0
(41)
0
(40)
0 (42)
0 (42)
PVP
0
(24)
0
(65)
0 (68)
0 (67)
5.6 (18)
0 (19)
0 (19)
DMSO
17 .6 (17)
The effect of warming rate was examined after cooling groups of 20 blastocysts at four different rates in the presence of 1 .5 M-DMSO .
An interaction
between cooling and warming rates was apparent (Table 5), with optimum warming
Vo1 .11,No . 22
Si~rvival of Flnbryo after Freezing
1076
rate becoming slower as the cooling rate was reduced . Maximum survival, which was 64 .7$, was obtained with a cooling rate of 0 .22°C/min and a warming rate of 12 °C/min .
After cooling at 0 .22° C/min and
warming at 1 .1 and 12 °C/min, 22 .5 and 64 .7$ of the blastocysts survived respactively .
The "exact probability" of such a large difference occurring by
chance in the absence of an effect of warming rate was found to be 0.033, which suggests that warming rate has a significant effect .
The exact test
was applied because of the small numbers of eggs in each treatment . TABLE 5 The effect of cooling rate and warming rate on the survival of early blastocysts frozen and thawed in the presence of 1.5 M-DMSO . The percentage which developed is shown, and, in parentheses, the number cultured . üarming rate °C/min 1.1
0.07
Cooling rate 0 .22
25 .0 (16)
12
0
(16)
3so
o
(ls)
°C/min 1 .2
4 .7
22 .5 (13)
0
(17)
0 (19)
64 .7 (17)
0
(19)
0 (18)
12 .s (ls)
o (ls)
o
(le)
Groups of 80 blastocysts were cooled at 0.22°C/min in the presence of 1.5 M-DMSO and warmed at 1 .1, 12 or 60°C/min.
The percentages which survived
following these three warming rates were 53 .8 (42 of 78), 85 .7 (66 of 77) and 64 .7 (44 of 68) respectively .
Comparisons of these waxing rates by Chi-
squared tests showed that 12 °C/min was the optimal rate and that 1 .1 and 60°C/ min were significantly different from each other.
(1 .1 v 12 °C/min P<0 .001 ;
12 v 60 °C/min P<0 .01) . Finally, when eight-cell eggs were cooled at 0.22°C/min and warmed at 12 °C/min, 65 .8$ developed (27 of 41) .
As 84 .0$ of untreated eggs developed
to blastocysts this represents a survival of 78 .4$ . DISCUSSION These experiments show clearly that a high proportion of mouse embryos, either at the eight-cell stage or as blastocysts, can survive freezing and storage in liquid nitrogen if they are suspended in medium containing DMSO .
Vo1 .11,No. 22
Survival of Fmbryo after Freezing
1077
So far viability has been demonstrated only by development in culture and it is important that this should be confirmed by transferring frozen and thawed embryos to recipient foster mothers .
Nevertheless, it seems probable that
these techniques will lead to the development of a practical method for the long term storage of embryos . The complete failure to obtain survival of blastocysts frozen and thawed in medium containing PVP in the present experiments confirms our previous observations and is again in contrast to the earlier report that survival was achieved when blastocysts xere held in this medium for 30 min in solid carbon dioxide (3) .
No valid explanation for this discrepâncy can be made .
Three observations of particular interest were made during these experimonts .
Firstly it xas noted that after freezing and thawing the cells in each
egg were usually either all damaged or all normal in appearance .
Furthermore,
similar proportions of eight-cell eggs and blastocysts survived freezing and thawing, which suggests that, in the main, freezing and thawing affected the embryos as a whole and not as individual cells xithin embryos .
Secondly, the
cooling rate which was found to be optimal was of the order expected either for very large cells or for cells which are, relatively, very impermeable to xater (17) .
Finally, it is very unusual for the optimum warming rate to be
as slow as 12 ° C/min .
In some circumstances, rapid warming of frozen plant
cells is found to be damaging (18), .and, in an isolated observation, elox warming of red blood cells was beneficial (19) .
Hoxever, with yeasts (11),
and hamster and mouse cells (12,15) rapid warming was never damaging and is some circumstances was clearly beneficial . Embryos at both of these stages of development are contained in a mucoprotein envelope, the zone pellucida, which has a diameter of approximately 100u, and it is possible that it is the presence of the zona which determines the optimum treatment.
Fihen the samples were seeded and ice formed, the
fluid outside the zona would have become hypertonic and equilibrium could only have been restored either by entry of solutes or by removal of water through
Survival of Fmbryo after Freezing
1078
the zone .
Vo1.11,No . 22
As the zones is relatively inflexible and unlikely to shrink, it
seems probable that movement of solutes restored equilibrium.
When the
cooling rate was sufficiently slow, ice would have been unlikely to form within the cells or the perivitelline space, as equilibrium could have been maintained by movement of solutes .
If such samples were warmed rapidly the
cells would have been exposed to a hypertonic environment at 20° C, whereas if the warming rate was slow, equilibrium could have been maintained by the loss of excess solutes from the perivitelline apace .
Thus, it is suggested that
because it determines the cooling rate at which ice formation in the perivitelline space or the cells becomes likely, it is the presence of the zone pellucida which determines that the optimum cooling and warming rates will be very slow . It has been established with a Chinese hamster cell that survival during freezing and thawing varies with the otage of the cell-cycle (20), and it is possible that the small number of dead cells which were observed in blastocysts which were fixed and stained after being frozen and thawed were those which were at a particularly sensitive phase of the cell-cycle when they were frozen and thawed . The practical value of techniques for long term storage of the embryos of large domestic animals has been stressed recently (21) .
Such techniques would
greatly facilitate the international exohange of genetically valuable livestock and could also provide a reservoir of embryos for use in the production of twins in cattle by a non-surgical transfer technique . I also I gratefully acknowledge receipt of a Milk Marketing Hoard Fellowship . wish to thank Dr . C. Polge for the provision of faciliti®s and for his advice, Dr . J. Ferrant for his advice during these experiments, Mr . D.E . Welters for advice on statistical analyses and Mr . K .T . Elsome for skilled technical assistance .
Vo1.11,No. 22
~
Sbrri~al of Fmbryo dta Freezing
1079
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