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Fast freezing of cow embryos in French straws with an automatic program
THERIOGENOLOGY
FAST
FREEZING WITH
A. Massip,
OF COW EMBRYOS IN FRENCH AN AUTOMATIC PROGRAM
STRAWS
P. Van der Zwalmen, C. Hanzen, F. Department of Reproduction Faculty of Veterinary Medicine 45, Rue des Veterinaires, 1070, Brussels, Belgium
Received
for
publication: Accepted:
Ectors
April 14, 1982 June 29,
1982
ABSTRACT
Cow embryos between day 6.5 and 9 were frozen in 1.5M DMSO in PBS at 2"C/min from seeding to -25°C before being plunged into liquid nitrogen directly or after 10 min at -25°C. Cooling rate from 20°C to -5°C was gOC/min.Seeding was induced automatically at -5'C by injection of liquid nitrogen vapour. Embryos were subsequently thawed by direct transfer to water at 20°C (group I) or at 37°C (group II). Survival was assessed by culture in vitro._and by transfer. after thawing (compared In group I, 35.7% were degenerated to 35.4% in group II). Survival rate after culture in vitro for 24h was not significantly different (48.3% vs 42-d hatching rate after 96h culture was quite similar (33.3% vs In group II, four pregnancies 34.4%). were obtained from 10 embryos transferred. Time at -25OC did not improve the results. Automatic seeding did not impair survival. These results show that the quality of the embryo is the determinant factor for survival after freezing and that the plastic straw is the most suitable vessel for freezing, storage and transfer of embryos. ACKNOWLEDGEMENTS The financial acknowledged.
SEPTEMBER
support
of
I.R.S.I.A.
1982 VOL. 18 NO. 3
is gratefully
325
THERIBGENOLOGY
INTRODUCTION The first freezing-thawing methods developed for cow embryos were laborious and time consuming (1,2). Although good survival rates were obtained (3), these methods were not practical on the farm. Recently, cow embryos have survived rapid thawing (360"C/min) provided that slow cooling at 0.3'C/min was terminated between -2O'C and -4O'C by direct transfer to liquid nitrogen (4,5,6). In this paper, we describe a very rapid method of freezing cow embryos in French straws with an entirely automatic program. In addition, we compare the effects of rapid thawing in a water bath at 20°C or at 37'C to the in vitro survival and development after transfer of day 6.5-day 9 cow embryos cooled at 2'C/min to -25°C and plunged immediately into liquid nitrogen or after 10 min at -25OC. MATERIAL
AND
METHODS
Embryos were collected non-surgically (7) between days 6.5 and 9 after onset of oestrus (day 0) from superovulated cows or heifers of mixed breeds as previously described (8). Stages of development of the embryos ranged from the compacted morula to the fully expanded and hatched blastocyst. All the embryos that were not degenerated or retarded were frozen. Freezing-thawing
method
1) The embryos were 1.5M DMSO in PBS
equilibrated at room by steps (9).
temperature
with
2) Two to three embryos were then aspirated in "mini-straws" (Cassou) within 100 ~1 of freezing medium between two very small air bubbles(l0) to avoid precipitous expulsion of plugs on thawing. 3) The straws were placed vertically in the cooling chamber of a programmable biological freezer (Minicool, Air Liquide) slightly modified for straws (Fig.1). The outside and inside a control temperature in the freezer, was recorded by means of straw containing freezing medium, a platinum probe and a NiCr-Ni thermocouple, 0.5 mm in diameter. The cooling program was as follows: rate of 9'C/min, - from 20°C to -5OC at a cooling - at -5°C liquid nitrogen vapour was injected with maximum cooling power during 4 seconds to induce seeding automatically.
326
SEPTEMBER
1982 VOL. 18 NO. 3
THERIQGENQLOGY
-_cfc
,
LID TEMPERATURE DETECTING ELEMENT FREEZER STRAW HEATING
VESSEL CARRIER ELEMENT
LIQUID NITROGEN INJECTOR NOZZLE SOLENOID VALVE NITROGEN PIPELINE
Figure
1.
Cooling
equipment
(ivlinicool)
by the cooling chamber after liquid The me an + sd to reached injection was terminated was 11.12 + 1.15"C. nitrog en iapour this injection, the t" of the chamber increas;d a few After degrees; then a rapid cooling cycle of Z"C/min was initiated k! ii?zC;emperature directly to liquid transferred after
(-25°C) some samples were transferred nitrogen'and the remaining samples 10 min at -25°C (Fig.2).
4)
After variable periods either in a water bath at 37°C (group II).
5)
DMSO was removed in transferred in PBS.
Viability
six
of at
storage, the straws were rewarmed 20°C (group I) or in a water bath
steps
(9)
then
embryos
were
assays
Morphological evaluation was made in PBS. Degenerated embryos were discarded; the others were either cultured in a modified Krebs-Ringer solution supplemented with 20% heat-inactivated sheep serum (8) or transferred through a flanc incision to synchronized recipient heifers. Survival in vitro was assessed by expansion or reexpansion after 24h of culture and hatching after 96h. 2 The data were analysed using Chi .
SEPTEMBER
1982 VOL. 18 NO. 3
327
TMERIOGENOLOGY
liquid vapour
nitrogen injection
Figure
RESULTS
2. Profile of temperature changes in the cooling chamber.
AND
DISCUSSION
The results are presented in Table 1. The proportion of degenerated embryos in group I (35.7%)did not differ significantly from that in group II (35.4%). Reexpansion invitro was quite similar between the two.groups'(48.3% in group I vs 42.8% in aroup 111 as well as the orooortion of hatching embryos (33:3% vs 34.4%). Four calves were born from embryos in the second group. The fact that these calves were obtained from excellent day 6.5 compacted morulae recovered from a selected donor cow is noteworthy. Of eight embryos collected and frozen, seven survived after thawing and six were transferred resulting in four calves born. The remaining surviving embryo was cultured and reexpanded in vitro. This indicates that the quality of the embryo de=rmines its chances of survivalto freezing and also that a rigorous selection of embryos to be frozen is necessary. Such was not the case in our study. The results in Table 1 indicate that thawing temperature had no significant effect on subsequent survival of embryos. When ministraws are warmed in a water bath at 2O“C it takes seven seconds for ice to disappear; in a water bath at 37OC this takes five seconds. Due to the small volume, thin wall, and large surface area of the straw in contact with water, the temperature of the thawing bath does not seem to be crucial for the survival of embryos, so tap water could probably be used conveniently. Embryos did not appear to be susceptible to cold shock when cooled abruptly from 20°C to the temperature at which automatic seeding took place. Four embryos out of six thawed after automatic seeding in water at 20°C hatched in vitro. In experiments with mouse blastocysts also thawed after automatic seeding in a water bath at 20°C, 69/76 (90.8%)
328
SEPTEMBER
1982 VOL. 18 NO. 3
W
Z P
$ F
S
E
56
E
2
z
F!z
12/11
50148
hatched
zona
compared
enclosed
84182
34134
0
10
-
29/28
17117
0
* including with
zona
:
embryos
bath
that
before
34.4
22.2
50
after
freezing had reexpansed
embryos
36(42.8)
20(40)
33.3
33.3
96 h
24h culture.
10
9
1
4
4
0
4
4
0
0
0
0
Pregnant
at -25°C.
Transfered
and time
(%)* Hatching(%)**
at 37'C
16(47.1)
water
14(48.3)
l( 8.3)
13(76.5)
24 h
temperature
in culture
at ZO'C _I-
Development
bath
of thawing
Reexpansion
water
as hatched
43
25
18
Thawing
14
1
13
Cultured
enclosed
as well
29(35.4)
14(29.2)
15(44.1)
lO(35.7)
6(54.5)
4(23.5)
(%)
:
as a function
Thawing
embryos
Degenerated
thawed
embryos
of frozen
Frozen/thawed
Viability
10
** embryos
Total
1.
at -25°C
(min.)
Total
Time
Table
-
THERIOGENOLOGY reexpanded after 24h culture in vitro and 68.1 % hatched after 48h in culture compared with 63.1% of 111 untreated blastocysts (Massip and Van der Zwalmen, unpublished observations). There was no significant difference in the survival rate,regardless of the maximum temperature reached by the cooling chamber after liquid nitrogen vapour injection, probably because seeding occurred invariably and completely at about the same temperature (between -8°C and -1O'C as observed on some samples). Willadsen (11) has shown that neither rapid cooling to -7OC nor seeding between -7'C and -1O'C for samples containing sheep embryos frozen to temperatures between -2O'C and -3O'C was harmful. Renard et al, (12) obtained similar results with cooling procedures starting at -7'C or at 2O*C (3). Automatic seeding is only possible with straws for the reasons mentioned above (small volume,large surface of contact and thin wall of the straw). Cow embryos survived at a cooling rate of 2*C/min from seeding temperature to -25°C. Lehn-Jensen and Greve (5) reported a 71 % survival rate with embryos cooled at before transfer to liquid 0.3"C/min down to -25'C nitrogen and thawed in water at 37OC. for 10 min did not influence Holding samples at -25*C the results probably because this cooling rate of 2'C/min is slow enough to provide sufficient shrinkage of embryos to ensure protection against injury on thawing from liquid nitrogen. Stages of development of embryos frozen ranged from to hatched blastocysts. Eleven out of compacted morulae 13 hatched blastocysts reexpanded after 24h in vitro but were degenerated after tnree days of culture. Three were transferred immediately after thawing to three recipient heifers;one was in oestrus at day 21 of the cycle, two returned to oestrus after 38 and 35 days This indicates that hatched blastocysts respectively. of day 8.5 to day 9 are not succesfully preserved by this procedure. Plastic straws are now widely used for freezing cow embryos because they simplify both the freezing ,storage In this paper, and transfer procedures (9,10,13,14). we have shown that an entirely automatic program could be used and recently a method was developed which allows transplantation of the embryos immediately after thawing, without needing to remove the cryoprotectant (15). Thus freezing-thawing methods have become shorter and simpler Their practical use as well as reliable, and effective. in the field on a large scale is obviously imminent.
330
SEPTEMBER
1982 VOL. 18 NO. 3
THERI[OGENOLOGY REFERENCES 1.
Experiments on the Wilmut,I.& Rowson,L.E.A.: rature preservation of cow embryos.-Vet.Rec. 690 (1973).
low tempe-92:686-
2.
Willadsen,S.M.,Trounson,A.O.,Polge,C.TRowson,L.E.A. Newcomb,R.: Low temperature preservation of Egg Transfer in Cattle.Ed.Rowson,L.E.A Aa'the European Communities,Luxemburg,ll7-ii5
3.
Willadsen,S.M.,Polge,C. & Rowson,L.E.A.: The viability of deep frozen cow embryos.J.Reprod.Fert. -52:391398 (1978).
4.
Willadsen,S.M.,Polge,C. & Rowson,L.E.A.: In vitro In: Control of Reproduction storage of cattle embryos. in the Cow.Ed.Sreenan,J.M.,Commission of the European Communities,Luxemburg,428-436 (1978).
5.
Survival of cow blastocysts Lehn-Jensen,H. & Greve,T.: utilizing short freezing curves.Nord.Vet.Med. -33: 523-529 (1981).
6.
of cow blastoLehn-Jensen,H. & Greve,T .: The survival cysts frozen in 1.4 M glycerol after plunging between -15'C and -6OOC and rapid thawing.Theriogenology -17: 95 (1982).
7.
Newcomb,R., recovery of ( 1978).
8.
Time-lapse cinematographic Massip,A. & Mulnard,J.: analysis of hatching of normal and frozen-thawed J.Reprod.Fert. (1980). blastocysts.-58: 475-478
9.
Massip,A., Vanderzwalmen,P.,Ectors,F.,De Coster,R., D'Ieteren, G. & Hanzen,C.: Deep freezing of cattle embryos in glass ampules or French straws.Theriogenology ( 1979). -12: 79-84
& cow eggs. Commission (1976).
Christie,W.B. & Rowson,L.E.A. : Non surgica bovine embryos.Vet.Rec. 102:414-417
cow
lO.Massip,A.,Ectors,F.,De Coster,R.,D'Ieteren,G.,Hanzen, C.,Vanderzwalmen,P. & Defonseca,M.: Deep-freezing of cattle embryos in French straws.9th Int.Congr. Anim. Vol. III ,Madrid, Reprod.& A.1 .,Proceedings 462, (abstr.) (1980). ll.Willadsen, S.M.: Factors affecting the survival of sheep embryos during deep-freezing and thawing. z : :t~; ;;;ezing of Mammalian Embryos (Ciba Found.Symp.), . Elsevier Excerpta Medica, Amsterdam(l977).
SEPTEMBER
1982 VOL. 18 NO. 3
331
THERIOGENOLOGY
12.
Renard,J.P.,Ozil,J.P. of deep frozen cattle 311-320 (1981).
13.
Lehn-Jensen,H.: Bovine egg transplantation tion of embryos.Nord.Vet.Med. -32:523-532
14.
Nieman,Von H.,Doepke,H.H.,Sacher,B. & Schilling,E.: Tiefgefrieren von Rinderembryonen in Plastikstraws mit AusverdUnnung des Gefrierschutzmittels durch Sucrose.Zuchthyg. 16: 201-205 (1981). -
15.
Renard,J.P.,Heyman,Y. & Ozi1,J.P.: Congelation de l'embryon bovin: une nouvelle methode de decongelation pour le transfert cervical d'embryons conditionnes une seule fois en paillettes.Ann.Mgd.Vet.,l26: 23-32 (1982).
332
& Heyman,Y.: Cervical transfer embryos.Theriogenology 15:
SEPTEMBER
.Preserva(1980).
1982 VOL. 18 NO. 3
FAST
FREEZING WITH
A. Massip,
OF COW EMBRYOS IN FRENCH AN AUTOMATIC PROGRAM
STRAWS
P. Van der Zwalmen, C. Hanzen, F. Department of Reproduction Faculty of Veterinary Medicine 45, Rue des Veterinaires, 1070, Brussels, Belgium
Received
for
publication: Accepted:
Ectors
April 14, 1982 June 29,
1982
ABSTRACT
Cow embryos between day 6.5 and 9 were frozen in 1.5M DMSO in PBS at 2"C/min from seeding to -25°C before being plunged into liquid nitrogen directly or after 10 min at -25°C. Cooling rate from 20°C to -5°C was gOC/min.Seeding was induced automatically at -5'C by injection of liquid nitrogen vapour. Embryos were subsequently thawed by direct transfer to water at 20°C (group I) or at 37°C (group II). Survival was assessed by culture in vitro._and by transfer. after thawing (compared In group I, 35.7% were degenerated to 35.4% in group II). Survival rate after culture in vitro for 24h was not significantly different (48.3% vs 42-d hatching rate after 96h culture was quite similar (33.3% vs In group II, four pregnancies 34.4%). were obtained from 10 embryos transferred. Time at -25OC did not improve the results. Automatic seeding did not impair survival. These results show that the quality of the embryo is the determinant factor for survival after freezing and that the plastic straw is the most suitable vessel for freezing, storage and transfer of embryos. ACKNOWLEDGEMENTS The financial acknowledged.
SEPTEMBER
support
of
I.R.S.I.A.
1982 VOL. 18 NO. 3
is gratefully
325
THERIBGENOLOGY
INTRODUCTION The first freezing-thawing methods developed for cow embryos were laborious and time consuming (1,2). Although good survival rates were obtained (3), these methods were not practical on the farm. Recently, cow embryos have survived rapid thawing (360"C/min) provided that slow cooling at 0.3'C/min was terminated between -2O'C and -4O'C by direct transfer to liquid nitrogen (4,5,6). In this paper, we describe a very rapid method of freezing cow embryos in French straws with an entirely automatic program. In addition, we compare the effects of rapid thawing in a water bath at 20°C or at 37'C to the in vitro survival and development after transfer of day 6.5-day 9 cow embryos cooled at 2'C/min to -25°C and plunged immediately into liquid nitrogen or after 10 min at -25OC. MATERIAL
AND
METHODS
Embryos were collected non-surgically (7) between days 6.5 and 9 after onset of oestrus (day 0) from superovulated cows or heifers of mixed breeds as previously described (8). Stages of development of the embryos ranged from the compacted morula to the fully expanded and hatched blastocyst. All the embryos that were not degenerated or retarded were frozen. Freezing-thawing
method
1) The embryos were 1.5M DMSO in PBS
equilibrated at room by steps (9).
temperature
with
2) Two to three embryos were then aspirated in "mini-straws" (Cassou) within 100 ~1 of freezing medium between two very small air bubbles(l0) to avoid precipitous expulsion of plugs on thawing. 3) The straws were placed vertically in the cooling chamber of a programmable biological freezer (Minicool, Air Liquide) slightly modified for straws (Fig.1). The outside and inside a control temperature in the freezer, was recorded by means of straw containing freezing medium, a platinum probe and a NiCr-Ni thermocouple, 0.5 mm in diameter. The cooling program was as follows: rate of 9'C/min, - from 20°C to -5OC at a cooling - at -5°C liquid nitrogen vapour was injected with maximum cooling power during 4 seconds to induce seeding automatically.
326
SEPTEMBER
1982 VOL. 18 NO. 3
THERIQGENQLOGY
-_cfc
,
LID TEMPERATURE DETECTING ELEMENT FREEZER STRAW HEATING
VESSEL CARRIER ELEMENT
LIQUID NITROGEN INJECTOR NOZZLE SOLENOID VALVE NITROGEN PIPELINE
Figure
1.
Cooling
equipment
(ivlinicool)
by the cooling chamber after liquid The me an + sd to reached injection was terminated was 11.12 + 1.15"C. nitrog en iapour this injection, the t" of the chamber increas;d a few After degrees; then a rapid cooling cycle of Z"C/min was initiated k! ii?zC;emperature directly to liquid transferred after
(-25°C) some samples were transferred nitrogen'and the remaining samples 10 min at -25°C (Fig.2).
4)
After variable periods either in a water bath at 37°C (group II).
5)
DMSO was removed in transferred in PBS.
Viability
six
of at
storage, the straws were rewarmed 20°C (group I) or in a water bath
steps
(9)
then
embryos
were
assays
Morphological evaluation was made in PBS. Degenerated embryos were discarded; the others were either cultured in a modified Krebs-Ringer solution supplemented with 20% heat-inactivated sheep serum (8) or transferred through a flanc incision to synchronized recipient heifers. Survival in vitro was assessed by expansion or reexpansion after 24h of culture and hatching after 96h. 2 The data were analysed using Chi .
SEPTEMBER
1982 VOL. 18 NO. 3
327
TMERIOGENOLOGY
liquid vapour
nitrogen injection
Figure
RESULTS
2. Profile of temperature changes in the cooling chamber.
AND
DISCUSSION
The results are presented in Table 1. The proportion of degenerated embryos in group I (35.7%)did not differ significantly from that in group II (35.4%). Reexpansion invitro was quite similar between the two.groups'(48.3% in group I vs 42.8% in aroup 111 as well as the orooortion of hatching embryos (33:3% vs 34.4%). Four calves were born from embryos in the second group. The fact that these calves were obtained from excellent day 6.5 compacted morulae recovered from a selected donor cow is noteworthy. Of eight embryos collected and frozen, seven survived after thawing and six were transferred resulting in four calves born. The remaining surviving embryo was cultured and reexpanded in vitro. This indicates that the quality of the embryo de=rmines its chances of survivalto freezing and also that a rigorous selection of embryos to be frozen is necessary. Such was not the case in our study. The results in Table 1 indicate that thawing temperature had no significant effect on subsequent survival of embryos. When ministraws are warmed in a water bath at 2O“C it takes seven seconds for ice to disappear; in a water bath at 37OC this takes five seconds. Due to the small volume, thin wall, and large surface area of the straw in contact with water, the temperature of the thawing bath does not seem to be crucial for the survival of embryos, so tap water could probably be used conveniently. Embryos did not appear to be susceptible to cold shock when cooled abruptly from 20°C to the temperature at which automatic seeding took place. Four embryos out of six thawed after automatic seeding in water at 20°C hatched in vitro. In experiments with mouse blastocysts also thawed after automatic seeding in a water bath at 20°C, 69/76 (90.8%)
328
SEPTEMBER
1982 VOL. 18 NO. 3
W
Z P
$ F
S
E
56
E
2
z
F!z
12/11
50148
hatched
zona
compared
enclosed
84182
34134
0
10
-
29/28
17117
0
* including with
zona
:
embryos
bath
that
before
34.4
22.2
50
after
freezing had reexpansed
embryos
36(42.8)
20(40)
33.3
33.3
96 h
24h culture.
10
9
1
4
4
0
4
4
0
0
0
0
Pregnant
at -25°C.
Transfered
and time
(%)* Hatching(%)**
at 37'C
16(47.1)
water
14(48.3)
l( 8.3)
13(76.5)
24 h
temperature
in culture
at ZO'C _I-
Development
bath
of thawing
Reexpansion
water
as hatched
43
25
18
Thawing
14
1
13
Cultured
enclosed
as well
29(35.4)
14(29.2)
15(44.1)
lO(35.7)
6(54.5)
4(23.5)
(%)
:
as a function
Thawing
embryos
Degenerated
thawed
embryos
of frozen
Frozen/thawed
Viability
10
** embryos
Total
1.
at -25°C
(min.)
Total
Time
Table
-
THERIOGENOLOGY reexpanded after 24h culture in vitro and 68.1 % hatched after 48h in culture compared with 63.1% of 111 untreated blastocysts (Massip and Van der Zwalmen, unpublished observations). There was no significant difference in the survival rate,regardless of the maximum temperature reached by the cooling chamber after liquid nitrogen vapour injection, probably because seeding occurred invariably and completely at about the same temperature (between -8°C and -1O'C as observed on some samples). Willadsen (11) has shown that neither rapid cooling to -7OC nor seeding between -7'C and -1O'C for samples containing sheep embryos frozen to temperatures between -2O'C and -3O'C was harmful. Renard et al, (12) obtained similar results with cooling procedures starting at -7'C or at 2O*C (3). Automatic seeding is only possible with straws for the reasons mentioned above (small volume,large surface of contact and thin wall of the straw). Cow embryos survived at a cooling rate of 2*C/min from seeding temperature to -25°C. Lehn-Jensen and Greve (5) reported a 71 % survival rate with embryos cooled at before transfer to liquid 0.3"C/min down to -25'C nitrogen and thawed in water at 37OC. for 10 min did not influence Holding samples at -25*C the results probably because this cooling rate of 2'C/min is slow enough to provide sufficient shrinkage of embryos to ensure protection against injury on thawing from liquid nitrogen. Stages of development of embryos frozen ranged from to hatched blastocysts. Eleven out of compacted morulae 13 hatched blastocysts reexpanded after 24h in vitro but were degenerated after tnree days of culture. Three were transferred immediately after thawing to three recipient heifers;one was in oestrus at day 21 of the cycle, two returned to oestrus after 38 and 35 days This indicates that hatched blastocysts respectively. of day 8.5 to day 9 are not succesfully preserved by this procedure. Plastic straws are now widely used for freezing cow embryos because they simplify both the freezing ,storage In this paper, and transfer procedures (9,10,13,14). we have shown that an entirely automatic program could be used and recently a method was developed which allows transplantation of the embryos immediately after thawing, without needing to remove the cryoprotectant (15). Thus freezing-thawing methods have become shorter and simpler Their practical use as well as reliable, and effective. in the field on a large scale is obviously imminent.
330
SEPTEMBER
1982 VOL. 18 NO. 3
THERI[OGENOLOGY REFERENCES 1.
Experiments on the Wilmut,I.& Rowson,L.E.A.: rature preservation of cow embryos.-Vet.Rec. 690 (1973).
low tempe-92:686-
2.
Willadsen,S.M.,Trounson,A.O.,Polge,C.TRowson,L.E.A. Newcomb,R.: Low temperature preservation of Egg Transfer in Cattle.Ed.Rowson,L.E.A Aa'the European Communities,Luxemburg,ll7-ii5
3.
Willadsen,S.M.,Polge,C. & Rowson,L.E.A.: The viability of deep frozen cow embryos.J.Reprod.Fert. -52:391398 (1978).
4.
Willadsen,S.M.,Polge,C. & Rowson,L.E.A.: In vitro In: Control of Reproduction storage of cattle embryos. in the Cow.Ed.Sreenan,J.M.,Commission of the European Communities,Luxemburg,428-436 (1978).
5.
Survival of cow blastocysts Lehn-Jensen,H. & Greve,T.: utilizing short freezing curves.Nord.Vet.Med. -33: 523-529 (1981).
6.
of cow blastoLehn-Jensen,H. & Greve,T .: The survival cysts frozen in 1.4 M glycerol after plunging between -15'C and -6OOC and rapid thawing.Theriogenology -17: 95 (1982).
7.
Newcomb,R., recovery of ( 1978).
8.
Time-lapse cinematographic Massip,A. & Mulnard,J.: analysis of hatching of normal and frozen-thawed J.Reprod.Fert. (1980). blastocysts.-58: 475-478
9.
Massip,A., Vanderzwalmen,P.,Ectors,F.,De Coster,R., D'Ieteren, G. & Hanzen,C.: Deep freezing of cattle embryos in glass ampules or French straws.Theriogenology ( 1979). -12: 79-84
& cow eggs. Commission (1976).
Christie,W.B. & Rowson,L.E.A. : Non surgica bovine embryos.Vet.Rec. 102:414-417
cow
lO.Massip,A.,Ectors,F.,De Coster,R.,D'Ieteren,G.,Hanzen, C.,Vanderzwalmen,P. & Defonseca,M.: Deep-freezing of cattle embryos in French straws.9th Int.Congr. Anim. Vol. III ,Madrid, Reprod.& A.1 .,Proceedings 462, (abstr.) (1980). ll.Willadsen, S.M.: Factors affecting the survival of sheep embryos during deep-freezing and thawing. z : :t~; ;;;ezing of Mammalian Embryos (Ciba Found.Symp.), . Elsevier Excerpta Medica, Amsterdam(l977).
SEPTEMBER
1982 VOL. 18 NO. 3
331
THERIOGENOLOGY
12.
Renard,J.P.,Ozil,J.P. of deep frozen cattle 311-320 (1981).
13.
Lehn-Jensen,H.: Bovine egg transplantation tion of embryos.Nord.Vet.Med. -32:523-532
14.
Nieman,Von H.,Doepke,H.H.,Sacher,B. & Schilling,E.: Tiefgefrieren von Rinderembryonen in Plastikstraws mit AusverdUnnung des Gefrierschutzmittels durch Sucrose.Zuchthyg. 16: 201-205 (1981). -
15.
Renard,J.P.,Heyman,Y. & Ozi1,J.P.: Congelation de l'embryon bovin: une nouvelle methode de decongelation pour le transfert cervical d'embryons conditionnes une seule fois en paillettes.Ann.Mgd.Vet.,l26: 23-32 (1982).
332
& Heyman,Y.: Cervical transfer embryos.Theriogenology 15:
SEPTEMBER
.Preserva(1980).
1982 VOL. 18 NO. 3