Note: Descriptions are shown in the official language in which they were submitted.
The prexent invention relates -to the
preservation by freezing of mammalian embryos. Such
embryos find applications in embryo transplantation.
There are known in the prior art small plastics
tubes~ called straws, containing spermatozoids in a suitable
liquid.
On the other hand, it is known that embryo
congelation requires a cryoprotector. However, removal of
this cryoprotector after thawing is necessary to ensure
subsequent v~ability of the embryo. It was previously
suggested to freeze a bovine embryo in a straw having a
structure different from that of the present invention,
wherein no provision was made to dilute the cryoprotector
after thawing~
The present invention provides a straw of a new
structure,whereby the cryoprotector can be removed without
manipulating the embryo.
In the straw according to the present invention,
the embryo i5 packed before freezing and said straw
is used as ~uch at time of transplantation.
The present invention provides a sealed straw
which cancels the need for plural handling o~ the embryo,
as requlred by the prior art. Thus, more precisely,one
single package is now necessary~viz.that used ~or the
2S setting withln the uterus of the receiver female.
The straw according to the invention makes it
possibl~ to use the frozen embryo,at the farm,without any
DA~ -2- 6740
handling on a microscopic scale. ~he immediately
available embryos may be stored in a portable cryogenlc
container . All prior art techniques required compulsorily
the previous resort to a biologist -~or, after thawing,
handling the embryo under a binocular lens.
The present invention has Por its obJect a straw
for the preservation by ~reezing o~ mammalian embryos
within a preservation medium capable oF being -frozen and
thawed and comprising a cryoprotect.ing substance (or
cryoprotector~which gets into the embryo cells9said straw
being characterized in that it is sealed at its ends
until the time of use in the mammal and in that the embryo
within the medium ~or preservatlon by freezing is
surrounded,respectiYely:
-(A~on the one hand,sequentially towards one end~
by (a) a gaseous mixture, tb) a culture medium,and (c)
a first sealing end plug,
~ (B)and on the other hand7sequentially towards
the other end,by ~d) a gas buble9 (e) a solution o~ at least
one water-miscible substance lowly~if at all,toxic at
ambient temperature to the embryo,whlch will not get into
the embryo cells but provides outslde sa~dembryo a
sufficient osmotic pre~sure to allow gradual di~usion
of the cryoprotector outwards oP the cells,the arrangement
being such that,upon thawing 9the embryo wlthin its
preservation medium ls directly contacted with said
solution,(~) another gas bubble and (g) a second sealing
end plug.
The present invention also relates to a straw
provided with an identi~ication rod for -the preservation
by freezing of mammalian embryos.
There exists nowadays a feed ~or means to identify
a Prozen embryo within a straw such as disclosed in the
present speci~ication.
The straws of the invention are stored before use in
liquld nitrogen. They usual:Ly bear on their body various
data requlred for identification,such as the reference
symbols of the organization where the embryos were
~rozen~the date o~ freezing,the breed and re~erences of
the donor ~emale.
Since such data are directly marked on the straw
body,an operator should for identitying purposes remove for
a short time the straw from the liquid nitrogen so as to
read the attendant in~orm3tion.
The embryo which is usually frozen within a
volume of 10-40 ~1 will undergo quite rapid rewarming as
soon as removed ~rom the liquid nitrogen. It is well known
that the rate of rewarming from - 196C to - 60C is
substantially of the order of 360C/min.
There~ore,direct reading on the straw gives rise
to an uncontrolled rewarming which promotes re-crystalliza-
tion o~ the frozen medium.
Such re-crystallization is all the more marked when
some amount of intracellular ice is present in the cells
(FARRANT, HEATHER and WALTER,1977-Effects of
interactions between cooling and rewarming conditions on
survival of cells.In "The freezing of Mammalian Emhryos,
Ciba Foundation Symposium.52, Elsevier, Excerpta Medica,
North Holland)~ Now, such is precisely the case with the
type of freezing now used for embryos
Thusgthere exists a problem as concerns
;dentification.
Moreover,there exists a problem as con,cerns the
calibration of the gas buble lying between the
cryoprotector contalning fractlon,and therefore the
embryo,and the fraction acting after thawing to dilute
the cryoprotector.
The present invention obviates to those problems
by providing a stra~ with a plug affording both precise
identi~ication of the embryo in said straw and means
for ¢alibrating the above mentioned gas bubble,whilst
permltting easier setting up of the straw.
According to the present invention,there is provided
a straw ~or the preservation by freezing o~ mammalian
embryos within a preservation medium capable of being
frozen and thawed and inoluding a cryoprotecting substance
(or cryoprotector)which gets into the embryo cells~ said
straw being sealed at its two ends un~il the time of use
in the mammal,the embryo within the medium ~or preservation
; by free2ing being respectively surrsunded:~A)on the one
~ ~ -5-
$~
hand,sequentially towards one end, by (a) a gaseous
mixture~ (b) a culture medium and (c) a first sealing
end plug, and
(B) on the other hand,sequentially towards the
other end, by (d) a gas bubble, (e~ a solution of at
least one water-miscible substance Iowly, if at all,
toxic at ambient temperature to the embryoJwhich will not
get into the embryo and provides outside said embryo a
suf~icient osmotic pressure to allow gradual diffusion
o~ the cryoprotector outwards of the cells~the
arrangement being such that1upon thawing7the embryo within
its preservation medium is directly contacted with said
solution,(f) another gas bubble and (g) a seaond sealing
end plug,wherein the second sealing end plug is a rod of
synthetic material whereof the body ls wider than that of
~aid straw and wherein that end of said rod which causes
the burst of the bubble rising,upon thawing,across the
medium fraction acting to dilute the cryoprotector,is
force-fitted through at least one flat into one end
of the straw9after filling thereof,to a depth which is
selected at will for precise calibration of said bubble.
The present invention also relates to the following
features, taken either singly or in every technically
po~sible combination thereof:
-the gaseous mixture (a) is selected among air,
nitrogen,blnary or ternary gaseous mixtures;
-the gaseous mixture ~a~ conslsts of nitrogen,
oxygen and carbon dioxide91n par-ticular in amoun-ts of 90
nitrogen~5~ oxygen and 5% carbon dioxide;
-the culture medium (b)is a medium for culture in
vitro with a carbonate or phosphate buffer, e.g. a
carbonate buffer essentially comprising glucose,mineral
salts and amino acids,or a medium with a pho~phate
buffer of the PBS type;
-the cryoprotector is glycerol,dimethylsulfoxide
or propanediol ;
~the gas bubble (d) between the embryo and the
solution (e~ consists of air,nitrogen or binary or
ternary gas mlxtures.
-the solution(e) contains sucrose and a salt
lS (phosphate or carbonate)huffer;
~the solution (e) contains polyvinylpyrrolidone
and a salt Iphosphate or carbonate)buffer;
-the normality o~ the salt buffer in solution (e)
is a function of the molarity of the substance present in
~he latter,the rule being that the buffer normality is all
the more low as the substance molarity is high;
-the gas bubble ~f) between the solution (e) and the
second plug (g) consists of air,nitrogen or binary or ternary
gas mixtures;
-the straw in frozen in a horizontal position;
-the relative volumes of the component elements for
an overall straw volume of` 500 microlitres is as follows
.. -7--
, -- . .
embryo 40 microlitres
gaseous mixture (a)50 microlitres
culture medium(b) 150 microlitres
gas bubble (d) between the
embryo and solution (e) 20 microlitres
solution (e~200 microlitres
gas bubbl0 (f) between
the solution and second
plug (g) 40 microlltres
-the end of the rod which causes bursting of the
bubble is in the shape of a portion of a sphere;
-the end of the rod which causes bursting of
the bubble is in the shape of a truncated cone;
~the end of the rod which causes bursting of the
bubble is in the shape of a cone;
-the shape of a truncated cone or a portion of a
sphere of the rod end is topped by a short point;
-the body of the rod includes symbols for
identifying the straw;
-the rod is made of lnJected plastics;
-the rod is made of vinyl polychlorlde;
~the force-fitted mounting of the rod is reinforced
by sub~ectlng the Junction area to a welding step.
The invention will be further illustrated by no way
of limitation with reference to the accompanying drawings
wherein:
~s~
Fig.l shows -the structure of a straw according
to the present invention bePore ~reezing;
Fig.2 shows the same straw as that in figure 1
upon thawing and cryoprotector dilution prior to application
of the straw to embryo transplantation;
Fig,3 illustrates the structure of a straw wi-th an
identification rod accordlng to the present invention
before freezing;
Fig.4 is a fragmentary view of one end of the rod
in the shape of a portion of a sphere;
Fi~.5 is a fragmentary view of one end of the rod In
the shape of a truncated cone;
Fig.6 is a fragmen~ary view of one end of the rod
in the shape of a cone.
Xn figure l,the straw according to the present
invention is generally designated at 1. It is stopped at
its two ends 13, 14 by a plug~ the two plugs being
designated 2 and 3 respectively. The first plug 2 is a
conventional plug consisting of cotton-wool impregnated
with polyvinyl alcohol. Such a plug was disclosed in
French patent 1 472 13~. The second plug 3 ma-y b~ a plug
identical with plug 2,but is not required to be so and may
be a mere polyvlnyl alcohol plug. A straw 1 of a 500-
microlltrescapdcity has a size of 13 cm for a
diameter of 2.7 centimetres. The embryo 4' lies in the
medium for preservation by freezing. This may be any
medium known for this purpose. The involved medium is
buffered and added with a cryoprotector 4 consisting,for
instance, of glycerol, dimethlsulfoxide or propanediol.
For further information upon such a medium for
preservation by freesing,those skilled in the art may refer
to the work: The Freezing of Mammalian Embryos,Ciba
Foundation Symposium,52,Elseview, North Holland, 1911,
and to the published work by Renard et al.1981:Teruigenology
15,113. By way of example,the concentration of cryoprotector
(glycerol)4 is about 1-1.5M. For a straw 1 of 500 microlitres,
the volume of embryo 4' in the medium for preservation by
freezing is 40 microlitres, the latter is surrounded
respectively on the side facing plug 2 by 50 microlitres of
a gaseous mixture 5.The gaseous mixture 5 may be
air, nitrogen or may comprise binary or ternary mixtures.
By way of example, the gaseous mixture 5 may consist of
nitrogen, oxygen and carbon dioxide, in particular in amounts
of 90% nitrogen, 5% oxygen and 5% carbon dioxide. Lying
between of gaseous mixture 5, having as mentioned above a
volume of subtantially 50 microlitres, and the plug 2 is
the culture medium 6. The culture medium 6 is any medium
suitable for culture in vitro. A specific example of such
a medium is a carbonate buffer medium marketed by the
firm API FRANCE under the designation "Milieu B2 INRA
MENEZO" and which essentially comprises glucose,mineral
salts,amino acids. Another medium 6 which may be used is
that available under the generic designation PBS
which is a phosphate buffer medium;a PBS medium is
especially described by Whittingham in "~ournal of
Reproduction and Fertility Supplement 14:7-21-19710
The medium for culture in vitro 6 permits both viability
of the embryo 4' and normal resumption of the metabolic
activity thereof upon thawing. On the side facing plug
3,the embryo 4' in the medium for preservation by freezing
containing the cryoprotector ~t is surrounded by a gas
bubble 7 of a 20~microlitre volume,then by a solution 8
of at least one water-miscible substance lowly toxic,
if at all ,at the ambient temperature to the embryo,which
does not get into the cells of the embryo 4' but provides
o~tside the embryo a sufficient osmotic pressure to allow
gradual diffusion of the cryoprotector 4 outwards of the
cells;said solution being designated by 8 and occyping,
in the example illustrated,a volume of 200 microlitres.
Finally,another gas bubble 9,of a 40-microlitre volume71ies
between solution 8 and plug 3.
The two gas bubbles 7 and 9 may consist of air,
nitrogen or comprise binary or ternary gas mixtures. The
preferred substance for solution 8 is sucrose Solution 8
contains,for example,sucrose and a ~phosphate or carbonate)
buffer having a normality selected as a function oF the
sucrose molarity. The buffer normality wlll be all the more
low as the sucrose molarity is high.A combination which
gave good results cons;sts of sUCrse 0.25Mfr a buffer
of 1 N. The sucrose may be replaced by polyvinylpyrrolidone,
the conditions set ~orth above remaining unchanged.
Solution 8 is a fraction permitting partial removal
of the cryoprotector 4 from the cells o-f embryo 4',0f
course~a tri~ling portion of cryoprotector 4 wlll anyway
subsist. The arrangement in the straw 1 according to the
present invention is such that~up thawing,the embryo 4'
in its preservation medium is clirectly contacted with
solution 8.
Ihe gas bubble 7 is a fraction which will vanish
during the rewarming process and allow Joining up o~ the
solution 8 and cryoprotector 4,while the gaseous mixture
5 acts to maintain until the transfer in utero the
separation between the cryoprotector and solution mixture
designated 11 and the culture medium 6.
Solution 8 does not get into the embryo cells but
acts by increasing the osmotic pressure of the medium
especially to limit the osmotic shocks on the cell
membranes during the passive di~fusion of the cryoprotector
4 outwards of the cells o~ embryo 4'.
Th~ various fractions in the above structure are
positioned by mere sucking with a l-ml syringe into the
straw 1 according to the present invention. Straw 1 is
sealed at its two ends 13,14 by means of two plugs 2 and 3
and the assembly as illustrated ln figure 1 is therea~ter
~rozen in horizontal position.
Figure 2 shows the thawing and dilution of the
cryoprotector 4 which in the example illustrated is glycerol
~12_
1~5 M.Upon thawing,the s-traw 1 is disposed in vertica1
position in a waker bath at a temperature of
substantially 37C designated 10 in figure 2.
Solution 8 ,which is less concentrated than the
solution of cryoprotector 4,is the first to begin melting
from the walls 15 of straw 1. Thus,at the thawing
temperature,solution 8 which has a concentration lower
than that of the medium Por preservation by freezing,is
the First to begin melting9this allowing the gas bubble 7J
from the very beginning of the temperature change attending
thawing,to rise within the straw l;the dimensions of said
gas bubble 7 are so selected that this rise will occur
gradually. The volume of the gaseous mixture 5,which i
of greater size than the gas bubble 7,remains practically
unchanged and maintains the separation between the
preservation medium and the culture medium 6.This affords
immediately upon thawing a sucrose~glycerol mixture 11.
The glycerolJsucrose volume ratio of 1:5 allows gradual
diffusion of the glycerol~ By alternatingly turning the
straw 1 up and down for 5-7 minutes,the embryo 4' is
allowed to flow all accross the liquid column9this
promoting homogeneous diffusion of the cryoprotector 4
Th~ s-traw 1 held in vertical position is cut at
its upper end,then mounted in a transplantation gun 12
2S A portion of the dilution medium ll,consisting of glycerol
and sucrose in the present example,is e~ected before
embryo 4' is set in place. Mixing with the culture
13
medium 6 is thus ensured durlng deposition of the embryo
41 in the uterus of the receiver female, In figure 2~
the same reference numerals are used to designate the
parts identical with those in figure 1.
In figures 3 to 6,the straw is generally designated
1 and its walls are desîgnated 15, The straw is stopped
at one end 13 thereof by a p.Lug 2,which is for instance
a polyvinyL alcohol plug, The embryo 4' lies in a
cryoprotector 4 and is surrounded respectively by a gaseous
mixture 5 and a gas bubhle 7, Lying between the gaseous
mixture 5 and plug 2 is the culture medium 6, A solution 8
of at least one water-miscihle substance lowly,i~ at all,
toxic at ambient temperature to the embryo9which does not
enter the cells o~ the embryo 4' but provides outside the
embryo a sufficient o~motic pressure to allow gradual
di~fusion of cryoprotector 4 outwards of the cells,is
provided hetween the gas bubble 7 and another gas bubble 9
separating said solution 8 from plug 3.
Plug 3 which is provided at the end 14 of straw 1
is a rod 31,made of plastics7having a body 32 wider
than that o~ straw 1. This rod lis crimped or force-fitted
at one o~ its ends 34 provided with at least one flat into
the end 14 of straw 1~ The end 34 of rod 31 in the shape
o~ a portion of a sphere 34a,a truncated cone 34b
: 25 or a cone 34c provided or not with a thin point 34d
14
facilitates upon thawing the bursting of bubble 7
when then latter comes to contact rod 31,this avolding
when the straw 1 is turned upside down a return motion of
bubble 7 within straw 1. Such a motion might disturb
the movement of embryo 4' accross the column
consisting of the mixture of ~ractions 4 and 8.Rod 31
is fitted~in after straw 1 has been fi~ed to a depth
which ls selected at will for precise calibration of
bubble 7. Due to a piston action derived from the
adJustable insertion of rod 31,bubble 7 may be calibrated
with a very high accuracy. Rod 31 includes symbols
required for ldenti~ying straw l;it is therefore possible,
according to the present invention,while retaining the
facility to have an identlfication mark directly on
straw l,to mark symbols on the plastics rod 31 which has
some thermal inertia and is fitted within the end 14
of straw 1. Read out only requires the removal of the
plastics rod 31 from the liquid nitrogen~whlle the
proper straw 1 is left within the liquid nitrogen.The
use of a color index Por the assembly body 32 of rod 31
end 14 allows a first identification for batches of
straws kept in a liquid nitrogen container. To keep the
length of the straw 1 plus rod 31 consistent with the
storing space in the various containers available at
present on the market,the overall length of straw 1 is
reduced as set forth hereaPter.
As mentioned above,the rod-and-plug assembly
3, 31 also ~acilitates the setting up of the straw. In
fact,to be able to rise in straw 1 upon thawing,bubble 7
should be of reduced volume, viz. 20 ~1 ~or 500 ~1
straws, 6 8 ~l for 250-~1 straws. Calibration o~ bubble 7
should be accurately done when sekting up straw 1. If the
volume of sucked air is slightly too small, the two
fractions are liable to become mixed and the step should
be repeated. The plastics rod 31 will,during insertion into
end 14 of straw l,compress the gaseous ~ractions during
assembly and the volume thereo~ will be reduced. This
decrease in volume which is dependent upon the depth
of penetration of rod 31 into straw 1 makes the setting up
easier, in that the operator may take an over-volume and
adJust as a ~inal step the volume o~ the air or gaseous
mixture fractions by varying the depth of insertion of rod
31 into str~,w 1.
The force~ ting or crimping of the plastics rod
31 into straw 1 may be strenghtened if so required by
subJecting the Junction area to a welding ac~ion. By such
procedure~for example,by means of a short welding pulse,the
Joining o~ the two plastics members can be enhanced so as
to render the straw 1 absolutely pilfer-proof.
Aocording to the present invention ,straws having
an internal diameter of 2.8 mm(corresponding to that of the
500-~1 straws)and straws having an internal diameter of
1.7 mm(corresponding to that of 250~1 straws) were produced.
i 16
Hereuncler are shown the relative volumes and
lengths of the component elements for a ].ow-diameter,viz:
250 ~ 1 straw.
Fraction Length Approximate
in mm
Embryo (4-4') 5 12
Gaseous mixture (5) 10 24
Gas bubble (7)between the embryo
~4-4')and the solution (8) 2~5 6
Sucrose solution (8) 25 55
Culture medium(6) 25 55
End air bubble (9~ 2.5
Overall stra~ (1) without plug (2) 70 160
Total straw (1) length
15 without rod (31) 87
Rod (31)1ength S5
Total length 142
The present invention is applicable to mammals. The
best results were obtained on bovine,ovine and caprine
embryos. However,it should be appreciated that the present
invention is not limited to application to ovine,bovine and
caprine embryos
The straw 1 of the present invention was used for
embryo transplantation.The results obtained are as
~o.llows:
From 30 embryos frozen,then cultivated in vitro,
24 or 48 hours after thawing,25 had a normal growth.This
figure represents a yield of 83.3%.
17
From 14 embryos -frozen~then directly -transplanted
a~er -thawing, 7 pregnancies ~o 55 days were obtained with
survival rates of 50%. The techniques used according to
the prior art only afforded a survival rate of 35%
The present inventlon lies in the particular
above described structure o~ the straw 1. It will be
appreciated that the plastics formlng the walls 15 is
a plastics conventionally use~d in strawstaccording to the
prior art,and is not within I;he scope of the present
invention.
Fînally,it is lmportant to note that the capacity
of straw l~of 500 microlitres, may vary and it is feasible
to produce straws which may have a different volume
such for example as 250~microlitre straws~
The presen~ invention also provides a straw
with an identification rod for the preservation by freezing
of mammalian embryos, said pla~tics rod 31 allowing
accurate identification of the embryo disposed in straw 1
and calibration at will of the gas bubble 7 whilst
~acilitating setting up of said straw 1. It should be
notlced that the rod 31 may be formed of any plastics
: which ~s oompatible with the fractlons in straw 1. By
way of illustration~sald rod 31 is made of vinyl polychlori-
de, but it should be understood that any s~milar material
is also suitahle The identification symbols on rod 31
may be produced in any suitable manner;for example,said
symbols may be printed using a rubber die.
18
The di-fference in width between the body 32 of
rod 31 and the body of straw 1 may be varied at will;
however it is preferred to have the body 32 of rod 31
only slightly wider than the body of straw 1.
The invention is not limited to the embodiments
shown and described and includes their various technical
equivalents and modifications within the purview of
those skilled in the art.
