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The effect of length of cryopreservation on the viability of bovine embryos in a commercial operation
THERIOGENOLOGY
THE EFFECT OF LENGTH OF CRYOPRESERVATION OF BOVINE EMBRYOS IN A COMMERCIAL
ON THE VIABILITY OPERATION
K. Hrugka, Jr. Embryo Transfer Center, JZD Agrokombinat 763 15 Slugovice, Czechoslovakia Received
for publication: Accepted:
October
6,
1990
July 11, 1991
ABSTRACT was obtained from A total of 2,232 bovine embryos 294 flushings at a commercial embryo transfer operation. The embryos were frozen in groups from individual flushings using 0.25-cc straws and a conventional freezing procedure The embryos were with glycerol as a cryoprotective agent. Sucrose was stored in liquid nitrogen for up to 28 months. used for the removal of glycerol after the thawing of embryos were then examined The thawed embryos. and 1,097 embryos (49%) with no apparent morphologically, The viability defects were used for subsequent transfer. flushes was of the thawed embryos from the individual the length of evaluated in relationship No correlationto (P > 0.1) was found cryopreservation. between the two parameters in embryos from superovulations This finding was with above and below average yields. further confirmed in a proportion of the embryos by the Thus, neither the typical evaluation of pregnancy rates. length of embryo storage in a commercial operation nor the success of superovulation influenced the survival rate of embryos after thawing based on morphological criteria and pregnancy rates. Key words:
cattle, embryo, cryopreservation, viability
Acknowledgements We thank Dr. Miroslav Dr. Vladimira Zelinova embryo handling.
morphology,
iak, Dr. Jaroslava Kyptova and for morphological assessment and
Present address: Department of Reproduction, University of Veterinary Science, Palacke'ho 1-3, 612 42 Brno, Czechoslovakia.
SEPTEMBER 1991 VOL. 36 NO. 3
477
THERIOGENOLOGY
INTRODUCTION Cryopreservation is an essential step in successful embryo transfer operations in cattle breeding (l-3). It has suggested that embryos kept frozen at the been temperature of liquid nitrogen (-196OC) should survive for centuries (4,5). obviously experimental results cannot prove that at present. Since cryopreserved semen cannot survive indefinitely and since a decrease in viability occurs after approximately a decade of storage in frozen liquid Kozumplik, pellets in nitrogen (J. personal communication, July 1990), the same may also be true for Despite the availability of frozen embryos. data describing the effects of various factors on the survival of frozen/thawed embryos (1,4,6-12), only a few authors have mentioned the actual length of cryopreservation (68,10,12). Due to methodological differences it is very difficult to compare these results or to apply them to a particular operation. The objective of our present study was to evaluate the effects of the length of cryopreservation on the post-thaw viability of bovine embryos, as assessed by a morphological examination and recipient pregnancy rates, under conditions of a commercial dairy cattle operation.
MATERIALS
AND METHODS
A total of 294 Holstein x Black and White dairy cows, up to 90 days post partum, were superovulated with p-FSH (Folicotropin SPOFA, Czechoslovakia; the total dose of 30 mg divided into four decreasing daily doses) between May 1986 and December 1988. The embryos were recovered on ovulation by a routine, Day 7 after nonsurgical The embryos gravitational flushing procedure (2). were separated by sedimentation (30 minutes) and evaluated A modified system of embryo grading (13) morphologically. was used. Embryos with more than 50% of an apparently normal, active cell mass were frozen. One-step equilibration (8) with 1.5 M glycerol was used prior to freezing. The embryos were packed in groups from the individual flushings into 0.25-cc straws in a buffered medium consisting of phosphate saline (PBS, Bioveta, Ivanovice, Czechoslovakia), 20% fetal calf serum University of Veterinary Science, Brno, (FCS, Czechoslovakia), and antibiotics. The straws were placed directly into the freezer chamber (Haake Qd Karlsruhe, Germany) and were set to a temperature of -6.5 C. After 15 minutes manual seeding was induced and 10 minutes later a programmed decrease in temperature was begun at the rate of -0.3°C/minute. After 30 minutes of equilibration at -32OC, the straws were plunged into liquid nitrogen.
478
SEPTEMBER 1991 VOL. 36 NO. 3
THERIOGENOLOGY
Storage time ranged from 1 to 849 days. The embryos were thawed in a water bath at 37OC (5 minutes). Glycerol was removed in a one-step procedure in a medium consisting of PBS, 20% FCS and 1.1 M sucrose (20 minutes). Only Grade 1 embryos (13) were considered suitable for transfer. The embryos were transferred transcervically (0.25-cc straws) to synchronized heifer recipients with the use of the Worrlein transfer catheter (Wbrrlein, Germany). Since this was a commercial facility many of the embryos were transferred in pairs ipsilaterally. Statistical evaluation was done "Basic employing Statistics," version 2.00 software (Veterinary Research and a PC AT 286. Institute, Brno, Czechoslovakia) The groups of embryos were divided according to the number of acquired from particular flushings: the aboveembryos average groups (~7 frozen embryos per flushing) and the below-average groups (=<7 frozen embryos per flushing). Correlation analysis was performed for the relationship between the percentage of suitable embryos after thawing and the length of cryopreservation; the rate of correlation and was expressed by the relevant coefficient its significance. Survivability in vivo was statistical evaluated by comparing the pregnancy rates in groups of recipients into which either single embryos or embryo pairs groups were categorized had transferred. The been according to the storage time of the embryos used.
RESULTS
Survival rates of embryos after thawing are summarized The rates were based on morphological in Figure 1. criteria with regard to the number of frozen embryos per flushing (above-average and below-average). On average, 7.59 embryos were frozen and 3.72 embryos were considered for transfer after thawing (2,232/294 and suitable The overall survival rate based on the 1,097/294). described evaluations of the embryos prior to and after cryopreservation was 49% (1,097/2,232). The distribution of the survival rates with regard to the length of cryopreservation was even, and no meaningful correlation was found. Pregnancy rates after embryo transfer of a portion of the 1,097 thawed embryos are summarized in Figure 2. Exact data on pregnancy rates in recipients, based on palpation per rectum 3 months after embryo transfer, were collected for 457 embryos. The remaining thawed embryos were from transferred both singly and in pairs the same collection, which made the interpretation of survivability in vivo difficult. A portion of the embryos was transferred in another project (14), and data on the pregnancies could not be reliably verified. SEPTEMBER 1991 VOL. 36 NO. 3
479
THERIOGENOLOGY
Viability afterthawing (<7embryosper f%ls~ wvvww v v v wvvw VW
Kuw t
V
0
im
all
300
400
600
WI
7m
(>7embryosper flushing) ImI
vv
V V
V
V
WV V
vV
V
V
V
w
V
V
v
Vv
v
vv V
V V
v w V v V Vv
v VV
V V V
V V
V
V
Length of cryopreservation Figure 1.
VV v
(days)
The viability of embryos after thawing in groups of 7 or less (above) and more than 7 (below) There was no frozen from individual flushings. correlation (P > 0.1) between the length and the , survival rate.
SEPTEMBER 1991VOL. 36 NO. 3
THERIOGENOLOGY
Pregnancy [after
transfer
rates
of
single
embryos)
90; se-Q1 .r( 78.h .H 8 60-e L 4r 0
SW40--
2
30--
$
20--
2
10-01
401-600 601-849 201-400 Days of cryopreseruatisn 0 nonpregnant pregnant
l-200 W
Pregnancy (after
transfer
rates of
embryo
pairs)
80 ;
70
0t
-4
l-200
I Figure 2.
201-400 401-600 601-849 Days of cryopreseruation pregnant 17 nonyregnnnt
of single rates after transfers The pregnancy embryos (above) and embryo pairs (below) from individual flushings groups of embryos from cryopreserved for different periods of time. The did not directly length of cryopreservation influence the pregnancy rates.
SEPTEMBER 1991VOL. 36 NO. 3
481
Both the transfer of single embryos and of embryo pairs were divided into four groups according to the length of the cryopreservation period (1 to 200 days, 201 to 400 No significant to 600 days, 601 to 849 days). days, 401 related to the length of in pregnancy rates decline cryopreservation was recorded (43, 50, 40 and 43% with single embryos and 45, 77.5, 52 and 31% with embryo pairs).
DISCUSSION It is not the objective of commercial cattle embryo transfer operations to keep embryos frozen for many years, since developments in genetic technologies may be quickly At present, cryopreservation is more important outdated. as a tool for making embryo recovery and embryo transfer short storage Consequently, independent of each other. (several months) is more frequently used than long storage (several years) periods. Adequate data on the effects of the length of storage viability are not in liquid nitrogen on the post-thaw Table 1 summarizes available data from our available. studies on the length of study and that of earlier However, only ranges were cryopreservation of embryos. attention was paid to the and no specific available, particular time periods. The time of cryopreservation and the survival rates of bovine embryos immediately after thawing
Table 1. Referegce study
No.of frozen Length of No.of thawed cryopreserembryos embryos vation (days)
1 6 7 8 10 12
1,770 736 335 117 623 240
Present
I:
482
study
2,234
1 28 43 1 240
to to to to to
270 210 241 240 630
1 to 849
Survival rate (%)
1,538 655 184 105 182 b 221
86.9 89.0 54.9 89.7
1,097
49.0
92.1
See text for reference numbers to the studies mentioned. Not all frozen embryos were thawed.
SEPTEMBER 1991 VOL. 36 NO. 3
THERIOGENOLOGY
With the exception of the experiments by Kusters (7), the overall survival rates were generally considerably The differences higher than those in our present study. can be attributed to different evaluation criteria for assessing the embryos as suitable for freezing. Frequently only Grade 1 embryos were frozen in the studies by other authors. The basis for determining threshold morphological quality for cryopreserving embryos differ according to the goals of the commercial embryo transfer operations. Our present investigation of groups of embryos from above-average and below-average superovulations was based on the idea that some donors produce more viable embryos than others. Although this "donor effect" has been proven experimentally (15), it is difficult to identify beforehand potential superior donors. No influence was found of the number of transferable embryos recovered per flushing on the survival rate of these embryos after thawing. concluded that the usual length of It can be cryopreservation of bovine embryos in liquid nitrogen in commercial embryo transfer operations does not affect the viability of embryos after thawing in vitro and in vivo.
REFERENCES 1.
Wright, straws.
Commercial freezing of bovine embryos J.M. Theriogenology Q:17-29 (1985).
Manual for embryo transfer. 2. Elsden, R.P. Journal the Society for Theriogenology B:l-63 (1987).
in of
The export of deep-frozen 3. Mahon, G.D. and Rawle, J.E. bovine embryos. Theriogenology =:21-35 (1987). 4. Hauschulte, H.M. Einfluss der Ausverdiinnungsmethode auf den Graviditatserfolg nach Transfer kryokonservierter Rinderembryonen. Dissertation, Justus-LiebigDniversitat Giessen, 1988. 5. Seidel, G.E. and Elsden, R.P. Dairy Cattle. Hoard's Dairyman WI, 1989, p. 76.
Embryo Transfer in Books, Ft. Atkinson,
6. Shea, B.F., McAlister, R.J. Janzen, R.E., and Freezing of bovine embryos: effects McDermand, D-P. of embryo quality, time from thawing to transfer and number frozen vial. Theriogenology =:205-212 per (1983). 7. Kiisters, G. Tiefgefrierversuche mit Rinderembryonen unter besonderer Berkksichtigung der Ausverdiinnungsmethoden. Dissertation, Tierarztliche Hochschule Hannover, 1983. SEPTEMBER 1991 VOL. 36 NO. 3
THERIOGENOLOGY
8.
Niemann, H. Freezing of bovine embryos: effects of a one-step addition of 1.4 M glycerol. Theriogenology =:369-379 (1985).
9.
Farrand, G.D., Elsden, R.P. and Seidel, G.E. Effect of slow cooling end point temperature on survival of frozen bovine embryos. J. Anim. Sci. =:460-465 (1985).
10.
Versuche zur Tiefgefrierkonservierung von Pavel, G. Rinderembryonen im One-Step Verfahren unter Vervendung eines Kryostaten. Dissertation, Tierarztliche Hochschule Hannover, 1985.
11.
Bielanski, A., Schneider, U., Pawlyshyn, V.P. and Mapletoft, R.J. Factors affecting survival of deep frozen bovine embryos in vitro: the effect of freezing container and method of removing cryoprotectant. Theriogenology =:429-437 (1986).
B. and Page, 12. Franks, G.C., Coley, S.L., Betterbed, R.D. The effect of freezer type, cryoprotectant, and processing methods on viability of frozen embryos. Theriogenology =:135-144 (1986). Bovine Embryo Grading. 13. Dorn, C.G. and Kraemer, D.C. Texas A & M University, College Station, TX, 1987, pp. 6-17. Export of frozen 14. Jiiizek, A. and Hrugka, K., Jr. bovine embryos and their transfers in Moldavia (in NdH chov 49:150-151 (1989). Czech). Untersuchung iiber Einflussfaktoren auf den 15. Camp, H. Embryotransfer Rind. Dissertation, Tier&ztliche Hochschule Hannover, 1989.
SEPTEMBER 1991 VOL. 36 NO. 3
THE EFFECT OF LENGTH OF CRYOPRESERVATION OF BOVINE EMBRYOS IN A COMMERCIAL
ON THE VIABILITY OPERATION
K. Hrugka, Jr. Embryo Transfer Center, JZD Agrokombinat 763 15 Slugovice, Czechoslovakia Received
for publication: Accepted:
October
6,
1990
July 11, 1991
ABSTRACT was obtained from A total of 2,232 bovine embryos 294 flushings at a commercial embryo transfer operation. The embryos were frozen in groups from individual flushings using 0.25-cc straws and a conventional freezing procedure The embryos were with glycerol as a cryoprotective agent. Sucrose was stored in liquid nitrogen for up to 28 months. used for the removal of glycerol after the thawing of embryos were then examined The thawed embryos. and 1,097 embryos (49%) with no apparent morphologically, The viability defects were used for subsequent transfer. flushes was of the thawed embryos from the individual the length of evaluated in relationship No correlationto (P > 0.1) was found cryopreservation. between the two parameters in embryos from superovulations This finding was with above and below average yields. further confirmed in a proportion of the embryos by the Thus, neither the typical evaluation of pregnancy rates. length of embryo storage in a commercial operation nor the success of superovulation influenced the survival rate of embryos after thawing based on morphological criteria and pregnancy rates. Key words:
cattle, embryo, cryopreservation, viability
Acknowledgements We thank Dr. Miroslav Dr. Vladimira Zelinova embryo handling.
morphology,
iak, Dr. Jaroslava Kyptova and for morphological assessment and
Present address: Department of Reproduction, University of Veterinary Science, Palacke'ho 1-3, 612 42 Brno, Czechoslovakia.
SEPTEMBER 1991 VOL. 36 NO. 3
477
THERIOGENOLOGY
INTRODUCTION Cryopreservation is an essential step in successful embryo transfer operations in cattle breeding (l-3). It has suggested that embryos kept frozen at the been temperature of liquid nitrogen (-196OC) should survive for centuries (4,5). obviously experimental results cannot prove that at present. Since cryopreserved semen cannot survive indefinitely and since a decrease in viability occurs after approximately a decade of storage in frozen liquid Kozumplik, pellets in nitrogen (J. personal communication, July 1990), the same may also be true for Despite the availability of frozen embryos. data describing the effects of various factors on the survival of frozen/thawed embryos (1,4,6-12), only a few authors have mentioned the actual length of cryopreservation (68,10,12). Due to methodological differences it is very difficult to compare these results or to apply them to a particular operation. The objective of our present study was to evaluate the effects of the length of cryopreservation on the post-thaw viability of bovine embryos, as assessed by a morphological examination and recipient pregnancy rates, under conditions of a commercial dairy cattle operation.
MATERIALS
AND METHODS
A total of 294 Holstein x Black and White dairy cows, up to 90 days post partum, were superovulated with p-FSH (Folicotropin SPOFA, Czechoslovakia; the total dose of 30 mg divided into four decreasing daily doses) between May 1986 and December 1988. The embryos were recovered on ovulation by a routine, Day 7 after nonsurgical The embryos gravitational flushing procedure (2). were separated by sedimentation (30 minutes) and evaluated A modified system of embryo grading (13) morphologically. was used. Embryos with more than 50% of an apparently normal, active cell mass were frozen. One-step equilibration (8) with 1.5 M glycerol was used prior to freezing. The embryos were packed in groups from the individual flushings into 0.25-cc straws in a buffered medium consisting of phosphate saline (PBS, Bioveta, Ivanovice, Czechoslovakia), 20% fetal calf serum University of Veterinary Science, Brno, (FCS, Czechoslovakia), and antibiotics. The straws were placed directly into the freezer chamber (Haake Qd Karlsruhe, Germany) and were set to a temperature of -6.5 C. After 15 minutes manual seeding was induced and 10 minutes later a programmed decrease in temperature was begun at the rate of -0.3°C/minute. After 30 minutes of equilibration at -32OC, the straws were plunged into liquid nitrogen.
478
SEPTEMBER 1991 VOL. 36 NO. 3
THERIOGENOLOGY
Storage time ranged from 1 to 849 days. The embryos were thawed in a water bath at 37OC (5 minutes). Glycerol was removed in a one-step procedure in a medium consisting of PBS, 20% FCS and 1.1 M sucrose (20 minutes). Only Grade 1 embryos (13) were considered suitable for transfer. The embryos were transferred transcervically (0.25-cc straws) to synchronized heifer recipients with the use of the Worrlein transfer catheter (Wbrrlein, Germany). Since this was a commercial facility many of the embryos were transferred in pairs ipsilaterally. Statistical evaluation was done "Basic employing Statistics," version 2.00 software (Veterinary Research and a PC AT 286. Institute, Brno, Czechoslovakia) The groups of embryos were divided according to the number of acquired from particular flushings: the aboveembryos average groups (~7 frozen embryos per flushing) and the below-average groups (=<7 frozen embryos per flushing). Correlation analysis was performed for the relationship between the percentage of suitable embryos after thawing and the length of cryopreservation; the rate of correlation and was expressed by the relevant coefficient its significance. Survivability in vivo was statistical evaluated by comparing the pregnancy rates in groups of recipients into which either single embryos or embryo pairs groups were categorized had transferred. The been according to the storage time of the embryos used.
RESULTS
Survival rates of embryos after thawing are summarized The rates were based on morphological in Figure 1. criteria with regard to the number of frozen embryos per flushing (above-average and below-average). On average, 7.59 embryos were frozen and 3.72 embryos were considered for transfer after thawing (2,232/294 and suitable The overall survival rate based on the 1,097/294). described evaluations of the embryos prior to and after cryopreservation was 49% (1,097/2,232). The distribution of the survival rates with regard to the length of cryopreservation was even, and no meaningful correlation was found. Pregnancy rates after embryo transfer of a portion of the 1,097 thawed embryos are summarized in Figure 2. Exact data on pregnancy rates in recipients, based on palpation per rectum 3 months after embryo transfer, were collected for 457 embryos. The remaining thawed embryos were from transferred both singly and in pairs the same collection, which made the interpretation of survivability in vivo difficult. A portion of the embryos was transferred in another project (14), and data on the pregnancies could not be reliably verified. SEPTEMBER 1991 VOL. 36 NO. 3
479
THERIOGENOLOGY
Viability afterthawing (<7embryosper f%ls~ wvvww v v v wvvw VW
Kuw t
V
0
im
all
300
400
600
WI
7m
(>7embryosper flushing) ImI
vv
V V
V
V
WV V
vV
V
V
V
w
V
V
v
Vv
v
vv V
V V
v w V v V Vv
v VV
V V V
V V
V
V
Length of cryopreservation Figure 1.
VV v
(days)
The viability of embryos after thawing in groups of 7 or less (above) and more than 7 (below) There was no frozen from individual flushings. correlation (P > 0.1) between the length and the , survival rate.
SEPTEMBER 1991VOL. 36 NO. 3
THERIOGENOLOGY
Pregnancy [after
transfer
rates
of
single
embryos)
90; se-Q1 .r( 78.h .H 8 60-e L 4r 0
SW40--
2
30--
$
20--
2
10-01
401-600 601-849 201-400 Days of cryopreseruatisn 0 nonpregnant pregnant
l-200 W
Pregnancy (after
transfer
rates of
embryo
pairs)
80 ;
70
0t
-4
l-200
I Figure 2.
201-400 401-600 601-849 Days of cryopreseruation pregnant 17 nonyregnnnt
of single rates after transfers The pregnancy embryos (above) and embryo pairs (below) from individual flushings groups of embryos from cryopreserved for different periods of time. The did not directly length of cryopreservation influence the pregnancy rates.
SEPTEMBER 1991VOL. 36 NO. 3
481
Both the transfer of single embryos and of embryo pairs were divided into four groups according to the length of the cryopreservation period (1 to 200 days, 201 to 400 No significant to 600 days, 601 to 849 days). days, 401 related to the length of in pregnancy rates decline cryopreservation was recorded (43, 50, 40 and 43% with single embryos and 45, 77.5, 52 and 31% with embryo pairs).
DISCUSSION It is not the objective of commercial cattle embryo transfer operations to keep embryos frozen for many years, since developments in genetic technologies may be quickly At present, cryopreservation is more important outdated. as a tool for making embryo recovery and embryo transfer short storage Consequently, independent of each other. (several months) is more frequently used than long storage (several years) periods. Adequate data on the effects of the length of storage viability are not in liquid nitrogen on the post-thaw Table 1 summarizes available data from our available. studies on the length of study and that of earlier However, only ranges were cryopreservation of embryos. attention was paid to the and no specific available, particular time periods. The time of cryopreservation and the survival rates of bovine embryos immediately after thawing
Table 1. Referegce study
No.of frozen Length of No.of thawed cryopreserembryos embryos vation (days)
1 6 7 8 10 12
1,770 736 335 117 623 240
Present
I:
482
study
2,234
1 28 43 1 240
to to to to to
270 210 241 240 630
1 to 849
Survival rate (%)
1,538 655 184 105 182 b 221
86.9 89.0 54.9 89.7
1,097
49.0
92.1
See text for reference numbers to the studies mentioned. Not all frozen embryos were thawed.
SEPTEMBER 1991 VOL. 36 NO. 3
THERIOGENOLOGY
With the exception of the experiments by Kusters (7), the overall survival rates were generally considerably The differences higher than those in our present study. can be attributed to different evaluation criteria for assessing the embryos as suitable for freezing. Frequently only Grade 1 embryos were frozen in the studies by other authors. The basis for determining threshold morphological quality for cryopreserving embryos differ according to the goals of the commercial embryo transfer operations. Our present investigation of groups of embryos from above-average and below-average superovulations was based on the idea that some donors produce more viable embryos than others. Although this "donor effect" has been proven experimentally (15), it is difficult to identify beforehand potential superior donors. No influence was found of the number of transferable embryos recovered per flushing on the survival rate of these embryos after thawing. concluded that the usual length of It can be cryopreservation of bovine embryos in liquid nitrogen in commercial embryo transfer operations does not affect the viability of embryos after thawing in vitro and in vivo.
REFERENCES 1.
Wright, straws.
Commercial freezing of bovine embryos J.M. Theriogenology Q:17-29 (1985).
Manual for embryo transfer. 2. Elsden, R.P. Journal the Society for Theriogenology B:l-63 (1987).
in of
The export of deep-frozen 3. Mahon, G.D. and Rawle, J.E. bovine embryos. Theriogenology =:21-35 (1987). 4. Hauschulte, H.M. Einfluss der Ausverdiinnungsmethode auf den Graviditatserfolg nach Transfer kryokonservierter Rinderembryonen. Dissertation, Justus-LiebigDniversitat Giessen, 1988. 5. Seidel, G.E. and Elsden, R.P. Dairy Cattle. Hoard's Dairyman WI, 1989, p. 76.
Embryo Transfer in Books, Ft. Atkinson,
6. Shea, B.F., McAlister, R.J. Janzen, R.E., and Freezing of bovine embryos: effects McDermand, D-P. of embryo quality, time from thawing to transfer and number frozen vial. Theriogenology =:205-212 per (1983). 7. Kiisters, G. Tiefgefrierversuche mit Rinderembryonen unter besonderer Berkksichtigung der Ausverdiinnungsmethoden. Dissertation, Tierarztliche Hochschule Hannover, 1983. SEPTEMBER 1991 VOL. 36 NO. 3
THERIOGENOLOGY
8.
Niemann, H. Freezing of bovine embryos: effects of a one-step addition of 1.4 M glycerol. Theriogenology =:369-379 (1985).
9.
Farrand, G.D., Elsden, R.P. and Seidel, G.E. Effect of slow cooling end point temperature on survival of frozen bovine embryos. J. Anim. Sci. =:460-465 (1985).
10.
Versuche zur Tiefgefrierkonservierung von Pavel, G. Rinderembryonen im One-Step Verfahren unter Vervendung eines Kryostaten. Dissertation, Tierarztliche Hochschule Hannover, 1985.
11.
Bielanski, A., Schneider, U., Pawlyshyn, V.P. and Mapletoft, R.J. Factors affecting survival of deep frozen bovine embryos in vitro: the effect of freezing container and method of removing cryoprotectant. Theriogenology =:429-437 (1986).
B. and Page, 12. Franks, G.C., Coley, S.L., Betterbed, R.D. The effect of freezer type, cryoprotectant, and processing methods on viability of frozen embryos. Theriogenology =:135-144 (1986). Bovine Embryo Grading. 13. Dorn, C.G. and Kraemer, D.C. Texas A & M University, College Station, TX, 1987, pp. 6-17. Export of frozen 14. Jiiizek, A. and Hrugka, K., Jr. bovine embryos and their transfers in Moldavia (in NdH chov 49:150-151 (1989). Czech). Untersuchung iiber Einflussfaktoren auf den 15. Camp, H. Embryotransfer Rind. Dissertation, Tier&ztliche Hochschule Hannover, 1989.
SEPTEMBER 1991 VOL. 36 NO. 3