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One-step sucrose dilution of frozen-thawed sheep embryos
THERIOCENOLOGY ONE-STEP SUCROSE DILUTION OF FROZEN-THAWED SHEEP EMBRYOS D. A. Merry, K. R. Bondiolia, R L. Allen and R. W. Wright, Jr. Deoartment of Animal Sciences College of Agriculture and Home Economics Washington State University Pullman, WA 99164-6332 Received for publication: May 11, 1984 Accepted: August 6, 1984 ABSTRACT Sheep embryos of the late morula to early blastocyst stage were frozen, thawed and cultured to test several sucrose solutions for post-thaw dilution of the cryoprotective agent glycerol. Ewes of mixed breeding were superovulated and embryos were flushed from the uterus either surgically or at slaughter 5 d after estrus. Fifty-eight embryos were pooled in microdrops of modified Dulbecco's phosphate buffered saline (MDPBS) then randomly divided into four treatments. A 2 x 2 factorial design was used to compare 0.25 M sucrose in MDPBS as an in-straw cryoprotectant dilution with a standard step-wise dilution procedure within standard fast and slow freeze-thaw systems. After storage in liquid nitrogen for 6 to 8 d, the embryos were thawed and the cryoprotectant (1.4 M glycerol) removed before culture in microdrops of modified synthetic oviduct fluid under paraffin oil in water-saturated 5% CO in air atmosphere at 37 C. No significant interaction was found be?ween the freeze-thaw procedure and cryoprotectant + dilution procedures. Embryos in the fast freeze-thaw procedure had a mean development score of 1.3 + 0.3 and those in the slow freeze-thaw procedure had a mean score of 1.2 + 0.3. The mean development score 2.0 f 0.3 for the standard dilution procedure was superior (P
Frozen, thawed, sucrose, sheep, embryos.
Acknowledgements Scientific Paper No. 6512. College of Agriculture and Home Economics Research Center, Washington State Univ., Pullman. Project No. 0313. Dept. of Anim. Sci. Medroxyprogesterone acetate pessaries kindly supplied by the Upjohn Co. Kalamazoo, MI. aPresent addres;: Granada Embryo Transfer, Route 1, Box 99A, Marquez, TX 77865.
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THERIOGENOLOGY INTRODUCTION The ability to store embryos of many domestic animals in liquid nitrogen for indefinite periods has improved the practicality of embryo transfer. Eight-cell to morula stage mouse embryos were the first embryos to be successfully frozen (1, 2). This was soon followed by the first live calf produced from a frozen-thawed embryo (3). Since then, freezing methods, cryoprotective agents, embryo freezing containers and other factors that have an effect on frozen-thawed embryo survival have been investigated for both laboratory and domestic species. Two freeze-thaw procedures are now commonly used for mammalian embryos. The first is a slow freezing system that requires carefully controlled slow thawing rates not easily duplicated under field conditions (4, 5). The second is a fast freezing system that requires fast thawing rates and is readily adaptable to field use. These systems have been shown to protect cow, sheep and mouse embryo viability equally well (6-8). One of the major limitations to the practicality of transfering frozen embryos in the field is the requirement that the cryoprotectant be removed from the embryo in a series of dilutions before transfer (5, 9). A one-step sucrose dilution system has recently been proposed (10-12) in which the cryoprotectant can be removed using a sucrose solution without removing the embryo from the freezing container. Observation and manipulation of the embryos before transfering is not necessary with this system. Early results indicate sucrose dilution to be as effective for removing cryoprotectant as standard dilution systems. The cow embryo has been used for much of this initial research with sucrose dilution because of the economic impact of this species. Similar research has also been done with mouse embryos (13). To date, there have been no reports on applying the one-step sucrose dilution system to frozen-thawed ovine embryos. The objectives of this study were: (1) to compare the success of sucrose and standard cryoprotectant dilution procedures in protecting sheep embryos viability; (2) to compare the success of the two standard freeze-thaw procedures protecting the sheep embryos viability; (3) to test for any interaction between the dilution and freezing procedures we used. MATERIALS
AN0 METHODS
Twenty-one cull ewes of mixed breeding were used as embryo donors. The majority of these ewes had been previously superovulated at least once. Ewes were superovulated with follicle stimulating hormone (FSH) after estrus synchronization with medoxyprogesterone acetate pessaries (14), then bred to at least two different rams. Fifty-eight embryos were collected from the uteri either at slaughter The reproductive tracts were (seven ewes) or surgery (14 ewes). exteriorized through a midline incision for the surgical collections. Flushing medium (60 ml to 90 ml) was injected at the base of each horn The flushing and collected at the ostium abdomenalie of the oviduct.
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medium used was modified Dulbecco's phosphate buffered saline (1) because this medium would also be used for the freezing procedures. The embryos were collected on three separate days. Only compact morulae and early blastocysts were used for freezing. Fifty-six embryos were morulae and two were very early blastocysts. The embryos were pooled and randomly assigned to one of four treatment groups. A 2 x 2 factorial design was used to compare two methods of cryoprotectant removal after a slow or fast standard freeze-thaw program. Fifty-eight embryos were assigned to four treatments as follows: 15 embryos in a slow freeze-thaw program with 0.25 M sucrose dilution; 14 embryos in a slow freeze-thaw program with standard step-wise dilution; 15 embryos in a fast freeze-thaw program with 0.25 M sucrose dilution; 14 embryos in a fast freeze-thaw program with standard step-wise dilution. For all treatments the embryos were added an increasing concentration of glycerol in three steps until a final concentration of 1.4 M: 0.47 M glycerol in modified Dulbecco's phosphate buffered saline MDPBS (10 min); 0.93 M glycerol in MDPBS (10 min); 1.4 M glycerol in MDPBS (30 min). After 15 min in the 1.4 M glycerol two or three embryos were loaded into the cryoprotectant of than appropriately loaded 0.5-ml French straw. Embryos for normal dilution were placed into straws loaded with approximately 400 pl of 1.4 M glycerol in MDPBS. Embryos for the sucrose dilution were placed into small drops (approximately 18 pl) of 1.4 M glycerol in MDPBS that were separated from a large volume (approximately 300 pl) of 0.25 M sucrose in MDPBS by an air bubble (figure 1). Straws were placed into their assigned freezing program when the 30 min to exposure time 1.4 M glycerol was complete. The slow freezing system used was essentially the same as that described by The embryos were cooled from Willadsen (4) and Willadsen et al., (5). room temperature to -7 C at 1 C/min. During a Z-min hold at this temperature, ice crystals were induced to form in the freezing medium with supercooled forceps (seeding). Following seeding, embryos were cooled at a rate of 0.3 C/min and at -60 C the straws were plunged into liquid nitrogen (-196 C). The fast freezing program was also essentially the same as one described by Willadsen (7). After storing the straws in liquid nitrogen for 6 d and 8 d, they were removed and thawed by the appropriate treatment method. Slow-frozen straws were placed in a -100 C atmosphere, warmed to -10 C Fast-frozen at 10 C/min and plunged into a 37 C water bath for 1 min. straw method were plunged directly from liquid nitrogen into a 37 C water bath for 1 min. After thawing, the normal-dilution straws were removed from the Each water bath and allowed to cool at room temperature for 2 min. After 3 min, the straw was then drained into a depression plate. embryos were placed in 1.17 M glycerol (10 min), 0.93 M glycerol (10 min), 0.70 M glycerol (10 min), 0.47 M glycerol (10 min), 0.23 M glycerol (10 min) and finally MDPBS (2 x 10 min each). All glYCerO1 The sucrose dilution straws were alSO concentrations were in MDPBS.
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Small drop of cryoprotectant -Small
air space
Large volume of sucrose
Sucrose dilutent to seal plug
Figure
1.
Diagrammatic representation of 0.5-ml French straw loaded with cryoprotectant and sucrose solutions for the one-step dilution procedure.
The air bubble allowed to cool at room temperature for 2 min. separating the embryos in 1.4 M glycerol in MDPBS from the 0.25 M sucrose in MDPBS was dislodged by tapping gently. Each straw was then drained into a depression plate and after 15 min the embryos were washed twice in MDPBS (10 min each). After cryoprotectant dilution and washing in MDPBS, all embryos were washed twice in modified synthetic oviduct fluid (10 min each). K. R. Bondioli (unpublished observation) found that sheep embryos developed well in synthetic oviduct fluid modified by replacing the 3.2% bovine serum albumin with fetal calf serum. The modification Two or used here employed 10% heat inactivated newborn lamb serum. three embryos were placed in each microdrop of modified synthetic oviduct fluid under paraffin oil at 37 C, in water-saturated 5% CO2 in air atmosphere (15).
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T'HERIOGENOLOGY To allow statistical treatment of data by analysis of variance the observed development of the embryos was coded as follows: 0 - no development; 1 - blastocyst; 2 - expanded blastocyst; 3 - hatching blastocyst; 4 - hatched blastocyst. Observations were made every 24 h and the maximum stage reached was recorded during 96 h of culture. This is a modification of a system used to find the cleavage index of bovine embryos in vitro (16). The data were analyzed by the general linear models procedure of the statistical analysis system (17) in the Washington State University computer. A separate trial with 18 embryos collected from seven ewes at a different time was also conducted. Three embryos were loaded into each of six sucrose dilution 0.5-ml French straws. The cryoprotectant was 1.4 M glycerol in MDPBS and the sucrose concentration was 1.0 M in MDPBS. All the straws were fast-frozen as previously described. Thawing and diluting procedures were performed in the same manner as for the fast-frozen 0.25 M sucrose dilution straws. Embryo culture and viability assessment were performed as described by Willadsen (7). RESULTS The number of embryos developed in vitro after each of the four freeze-thaw treatments is given in table 1. The proportion of embryos showing any development (received a score of 1 or more) is also given for each treatment. One embryo was not recovered from one straw in each of the two treatments using standard dilution. It could not be determined if this loss was due to a treatment effect, so these embryos were excluded from the calculations. Eighty percent of the slow-freeze standard dilution and 69% of the fast-freeze standard dilution treatments showed some development in vitro. Both the slow-freeze sucrose dilution and fast-freeze sucrose dilution treatments had only 33% of the embryos showing any development in vitro. TABLE 1.
EFFECT OF FREEZE-THAW AND CRYOPROTECTANT DILUTION PROCEDURES ON THE DEVELOPMENT OF SHEEP MORULAE IN VITRO
Freeze-thaw procedure
Cryoprotectant dilution procedure
Proportion Development scoresa 0 12 3 4
0 deve;oping,
bSlow Slow
Standard 0.25 M Sucrose
3 10
2 4
2 0
4 0
2 1
80 33
bFast Fast
Standard 0.25 M Sucrose
4 10
114 2
1
1
3 1
69 33
aDevelopment was scored at the cessation of culture as: 0 - no development; 1 - blastocyst; 2 - expanded blastocyst; 3 - hatching blastocyst; 4 - hatched blastocyst. b One embryo not recovered after thawing.
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THERIOGENOLOGY Because the interaction between the freeze-thaw procedure and the dilution procedure was not significant, the results were pooled for statistical analysis of the main effects. The average development score for each main treatment are given in table 2. The freeze-thaw procedures were not found to be significantly different. Embryos in the fast freeze-thaw procedure had a mean development score of 1.3 f 0.3 and those in the slow freeze-thaw procedure a mean score of 1.2 + 0.3. There was a highly significant (P 5 0.001) difference in the mean development scores for each dilution procedure. The 0.25 M sucrose dilution was inferior to the standard dilution procedure with development scores of 0.6 + 0.2 and 2.0 f 0.3, respectively.
TABLE
2.
EFFECT OF FREEZE-THAW AND CRYOPROTECTANT ON THE MEAN DEVELOPMENT OF SHEEP MORULAE
Freeze-thaw and Cryoprotectant dilution
Freeze-thaw
PROCEDURES
Development scorea, x + SE
embryos
procedure
Fast
28
1.4 + 0.3
Slow
28
1.2 f 0.3
Standard
26
2.0 * 0.3b
.25 M sucrose
30
0.6 f 0.2'
Cryoprotectant procedure
aFor
Total
DILUTION IN VITRO
explanation
"cValues
with
dilution
of development different
scores
superscripts
see table differ
1.
(PLO.001).
The same mean development for the standard dilution and 0.25 M sucrose dilution procedures are given in table 3, along with the mean development score for the separate 1.0 M sucrose dilution trial. Statistical comparisons of the 1.0 M sucrose procedure to the other two dilution procedures are not relevant because they were conducted on separate groups of embryos. A strictly visual comparison indicates that the 1.0 M sucrose dilution procedure may be marginally superior to the 0.25 M sucrose dilution. However, this 1.0 M sucrose dilution The sucrose is not as effective as the standard dilution procedure. dilution embryos in general had less extruded cellular material than the dilution embrvos immediatelv after completing of the dilution procedure. Post-dilution embryo morphology was optimum with 1.0 M sucrose dilution procedures.
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TABLE
3.
EFFECT OF CRYOPROTECTANT OILUTION PROCEDURES DEVELOPMENT OF FROZEN-THAWED SHEEP MORULAE
Cryoprotectant procedure
dilution Total
ON THE
IN VITRO
Development scorea, x f SE
embryos
Standard
26
2.0 f 0.3
.25 M Sucrose
30
0.6 + 0.2
1.00
M Sucrose
16
1.0 f 0.3
aFor
explanation
One to form varying embryos embryos as shown
of development
scores
1.
embryo in figure 2 appears to be vesiculating, but developed a hatched blastocyst. This vesiculated appearance with amounts of dead and extruded material was commonly seen in Nine out of the 28 that developed after freezing and thawing. (32%) in the fast-thaw treatment had cracked zone pellucidae in figure 2. No cracked zonae were observed in the slow-thaw
A
Figure
see table
2.
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One typical sheep embryo one typical sheep embryo Magnification 100X
1984 VOL. 22 NO. 4
B
with damaged zona pellucida in early development.
and
439
THERIOGENOLOCY procedure. development
Damage to the zona in vitro.
pellucida
did
not affect
embryo
DISCUSSION The fast and slow freeze-thaw methods used with the normal dilution procedures are virtually identical to procedures used in previous studies (4, 5, 7). The percentage of embryos developing in vitro from these procedures agrees with the results reported for frozen-thawed sheep embryos by Willadsen et al. (18) and Willadsen (4) and with a report by Polge and Willadsen (6), which suggests that the fast and slow freeze-thaw methods do not have different abilities to promote the in vitro development of sheep embryos. The 0.25 M sucrose in MDPBS dilution procedure was not found to be effective as a standard procedure in developing sheep embryos in vitro. There was also no significant interaction between the freeze-thaw procedure and the dilution procedure. The 1.0 M sucrose in MDPBS dilution procedure was not as effective as the standard dilution procedure. Due to the limited number of embryos employed, further experimentation with 1.0 M sucrose will be necessary. A. 0. Trounson (personal communication) has found sucrose concentrations in the range of 1.0 M to be effective for cryoprotectant dilution of sheep embryos. A number of cracked zona pellucidae were noted in the fast frozen-thawed embryos. Damage to the zona pellucida has not been shown to affect the viability of frozen-thawed embryos (7, 19). However, it does make the embryos more sensitive to disruption during manipulation. S. P. Leibo (personal communication) has indicated that thawing rates slower than those produced by a 37 C waterbath thaw will reduce zone damage. Kasai et al. (13) demonstrated that mouse embryos thawed at 360 C/min (37 C water bath) were more viable in vitro than embryos thawed at 25 C/min (room-temperature air thaw). Further study will be required to establish the optimum warming rate and the methods needed to reproduce it consistently in the field. After 96 h in culture, all the frozen-thawed embryos appeared to be dead or dying. Many embryos had numerous vesicles and extruded dead material. The embryos that appeared to be vesiculating were generally the only embryos to develop to at least the blastocyst stage. Many cells are killed by the freeze-thaw or dilution process, but the surviving cells may reform a blastocyst. Dead material is extruded from the living cell mass and can be seen as dark patches on the exterior of most blastocysts. The morphological appearance of embryos from the 0.25 M sucrose dilution was superior to that of embryos treated by the standard dilution procedure. The appearance of the 1.0 M sucrose-diluted embryos was superior to that of the 0.25 M sucrose-diluted embryos. The morphological evaluation of these embryos after cryoprotectant dilution does not correlate well with their ability to develop in vitro. Elsden et al. (20) noted that post-thaw morphological evaluation does not correlate well with in vivo development of frozen-thawed bovine embryos.
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THERIOGENOLOGY The one-step dilution system with a sucrose gradient appears to be a simple and efficient method to thaw embryos and is being used successfully with bovine embryos. However, this study suggests that ovine embryos have a more reduced viability in culture following sucrose dilution when compared with standard dilution techniques.
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THERIOGENOLOGY REFERENCES 1.
Whittingham, D.G. Survival of mouse embryos after freezing and thawing. Nature 233:125-126. (1971)
2.
Whittinghan, D.G., Leibo, S.P. and Mazur, P. Survival of mouse embryos frozen to -196 and -296 C. Science 178:411-414. (1972)
3.
Wilmut, I. and Rowson, L.E.A. The successful low-temperature preservation of mouse and cow embryos. J. Reprod. Fertil. a:252 (Abstr.). (1973)
4.
Willadsen, S.M. Deep freezing of sheep embryos. In: Elliott, K. and Whelan, J. (Eds) The Freezing of Mammalian Embryos, Ciba Fndn. Symp. No. 52 (new series). Elsevier/North Holland, Amsterdam, 1977, pp. 175-189.
5.
Willadsen, S.M., Polge, C. and Rowson, L.E. The viability of deep-frozen cow embryos. J. Reprod. Fertil. 52:391-393. (1978)
6.
Polge, C. and Willadesn, S.M. Freezing eggs and embryos of farm animals. Cryobiology 15:370-373. (1978)
7.
Willadsen, S.M., Polge, C. and Rowson, L.E. In vitro storage of cattle embryos. In: Control of Reproduction In the Cow. Commission of theTuropean Communities, The Hague, Netherlands, 1978, pp. 427-436.
8.
Whittingham, D.G., Wood, M., Farrant, J., Lee, H. and Halsey, J.A. Survival of frozen mouse embryos after rapid thawing from -196 C. J. Reprod. Fertil. !&:ll-21.
9.
Lehn-Jensen, H. bovine embryos.
Bovine egg transplantation. Preservation of Nord. Vet. Med. 2:523-532. (1980)
10.
Renard, J.-P., Heyman, Y. and Ozil, 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. Med. Vet. 126:23-32. (1982)
11.
Leibo, S.P. Field trial of one-step frozen bovine embryos transferred non-surgically. Theriogenology 19:139 (Abstr.). (1984)
12.
Renard, J.-P., Heyman, Y., Leymonie, P. and Plat, J.-C. Sucrose dilution: A technique for field transfer of bovine embryos frozen in the straw. Theriogenology -19:145 (Abstr.). (1983)
13.
Kasai, M., Niwa, K. and Iritani, A. Survival of mouse embryos frozen and thawed rapidly. J. Reprod. Fertil. 2:51-56. (1980)
14.
Wright, R.W., Jr., Bondioli, K., Grammer, J., Kuzan, F.and Menino, Jr., A. FSH or LH plus FSH superovulation in ewes following estrus synchronization with medroxprogesterone acetate pessaries. J. Anim. Sci. 52:115-118. (1981)
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15.
Brinster, R.L. A method for in vitro cultivation of mouse ova from two-cell to blastocyst. Exp. Cell. Res. 32:205-208. (1963)
16.
Wright, R.W., Jr., Anderson, G.B., Cupps. P.T. and Orost, M. Successful culture in vitro of bovine embryos to the blastocyst stage. Biol. Reprod. -14:157-162. (1976)
17.
Barr, J.J., Goodnight, J.H., Sall, J.P., Blair, W.H., Chilka, D.M., Council, K.A. and Helwig, J.T. SAS User's Guide, SAS Institute, Inc., Raleigh, NC, 1979, pp.
18.
Willadsen, S.M., Polge, C., Rowson, L.E. and Moor, R.M. Deep 46:151-154. (1976) freezing of sheep embryos. J. Reprod. Fertil. -
19.
Massip, A., Van der Zwalmen, P., Factors, F., DeCoster, R., D'Ieteren, G. and Hanzen, C. Deep freezing of cattle embryos in glass ampules of french straws. Theriogenology 12:79-84. (1979)
20.
Elsden, R.P., Seidel, Jr., G. E., Takeda, T. and Farrand, G.D. Field experiments with frozen-thawed bovine embryos transferred nonsurgically. Theriogenology 17:1-10. (1982)
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Frozen, thawed, sucrose, sheep, embryos.
Acknowledgements Scientific Paper No. 6512. College of Agriculture and Home Economics Research Center, Washington State Univ., Pullman. Project No. 0313. Dept. of Anim. Sci. Medroxyprogesterone acetate pessaries kindly supplied by the Upjohn Co. Kalamazoo, MI. aPresent addres;: Granada Embryo Transfer, Route 1, Box 99A, Marquez, TX 77865.
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THERIOGENOLOGY INTRODUCTION The ability to store embryos of many domestic animals in liquid nitrogen for indefinite periods has improved the practicality of embryo transfer. Eight-cell to morula stage mouse embryos were the first embryos to be successfully frozen (1, 2). This was soon followed by the first live calf produced from a frozen-thawed embryo (3). Since then, freezing methods, cryoprotective agents, embryo freezing containers and other factors that have an effect on frozen-thawed embryo survival have been investigated for both laboratory and domestic species. Two freeze-thaw procedures are now commonly used for mammalian embryos. The first is a slow freezing system that requires carefully controlled slow thawing rates not easily duplicated under field conditions (4, 5). The second is a fast freezing system that requires fast thawing rates and is readily adaptable to field use. These systems have been shown to protect cow, sheep and mouse embryo viability equally well (6-8). One of the major limitations to the practicality of transfering frozen embryos in the field is the requirement that the cryoprotectant be removed from the embryo in a series of dilutions before transfer (5, 9). A one-step sucrose dilution system has recently been proposed (10-12) in which the cryoprotectant can be removed using a sucrose solution without removing the embryo from the freezing container. Observation and manipulation of the embryos before transfering is not necessary with this system. Early results indicate sucrose dilution to be as effective for removing cryoprotectant as standard dilution systems. The cow embryo has been used for much of this initial research with sucrose dilution because of the economic impact of this species. Similar research has also been done with mouse embryos (13). To date, there have been no reports on applying the one-step sucrose dilution system to frozen-thawed ovine embryos. The objectives of this study were: (1) to compare the success of sucrose and standard cryoprotectant dilution procedures in protecting sheep embryos viability; (2) to compare the success of the two standard freeze-thaw procedures protecting the sheep embryos viability; (3) to test for any interaction between the dilution and freezing procedures we used. MATERIALS
AN0 METHODS
Twenty-one cull ewes of mixed breeding were used as embryo donors. The majority of these ewes had been previously superovulated at least once. Ewes were superovulated with follicle stimulating hormone (FSH) after estrus synchronization with medoxyprogesterone acetate pessaries (14), then bred to at least two different rams. Fifty-eight embryos were collected from the uteri either at slaughter The reproductive tracts were (seven ewes) or surgery (14 ewes). exteriorized through a midline incision for the surgical collections. Flushing medium (60 ml to 90 ml) was injected at the base of each horn The flushing and collected at the ostium abdomenalie of the oviduct.
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THERIOCENOLOGY
medium used was modified Dulbecco's phosphate buffered saline (1) because this medium would also be used for the freezing procedures. The embryos were collected on three separate days. Only compact morulae and early blastocysts were used for freezing. Fifty-six embryos were morulae and two were very early blastocysts. The embryos were pooled and randomly assigned to one of four treatment groups. A 2 x 2 factorial design was used to compare two methods of cryoprotectant removal after a slow or fast standard freeze-thaw program. Fifty-eight embryos were assigned to four treatments as follows: 15 embryos in a slow freeze-thaw program with 0.25 M sucrose dilution; 14 embryos in a slow freeze-thaw program with standard step-wise dilution; 15 embryos in a fast freeze-thaw program with 0.25 M sucrose dilution; 14 embryos in a fast freeze-thaw program with standard step-wise dilution. For all treatments the embryos were added an increasing concentration of glycerol in three steps until a final concentration of 1.4 M: 0.47 M glycerol in modified Dulbecco's phosphate buffered saline MDPBS (10 min); 0.93 M glycerol in MDPBS (10 min); 1.4 M glycerol in MDPBS (30 min). After 15 min in the 1.4 M glycerol two or three embryos were loaded into the cryoprotectant of than appropriately loaded 0.5-ml French straw. Embryos for normal dilution were placed into straws loaded with approximately 400 pl of 1.4 M glycerol in MDPBS. Embryos for the sucrose dilution were placed into small drops (approximately 18 pl) of 1.4 M glycerol in MDPBS that were separated from a large volume (approximately 300 pl) of 0.25 M sucrose in MDPBS by an air bubble (figure 1). Straws were placed into their assigned freezing program when the 30 min to exposure time 1.4 M glycerol was complete. The slow freezing system used was essentially the same as that described by The embryos were cooled from Willadsen (4) and Willadsen et al., (5). room temperature to -7 C at 1 C/min. During a Z-min hold at this temperature, ice crystals were induced to form in the freezing medium with supercooled forceps (seeding). Following seeding, embryos were cooled at a rate of 0.3 C/min and at -60 C the straws were plunged into liquid nitrogen (-196 C). The fast freezing program was also essentially the same as one described by Willadsen (7). After storing the straws in liquid nitrogen for 6 d and 8 d, they were removed and thawed by the appropriate treatment method. Slow-frozen straws were placed in a -100 C atmosphere, warmed to -10 C Fast-frozen at 10 C/min and plunged into a 37 C water bath for 1 min. straw method were plunged directly from liquid nitrogen into a 37 C water bath for 1 min. After thawing, the normal-dilution straws were removed from the Each water bath and allowed to cool at room temperature for 2 min. After 3 min, the straw was then drained into a depression plate. embryos were placed in 1.17 M glycerol (10 min), 0.93 M glycerol (10 min), 0.70 M glycerol (10 min), 0.47 M glycerol (10 min), 0.23 M glycerol (10 min) and finally MDPBS (2 x 10 min each). All glYCerO1 The sucrose dilution straws were alSO concentrations were in MDPBS.
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THERIOGENOLOGY
Small drop of cryoprotectant -Small
air space
Large volume of sucrose
Sucrose dilutent to seal plug
Figure
1.
Diagrammatic representation of 0.5-ml French straw loaded with cryoprotectant and sucrose solutions for the one-step dilution procedure.
The air bubble allowed to cool at room temperature for 2 min. separating the embryos in 1.4 M glycerol in MDPBS from the 0.25 M sucrose in MDPBS was dislodged by tapping gently. Each straw was then drained into a depression plate and after 15 min the embryos were washed twice in MDPBS (10 min each). After cryoprotectant dilution and washing in MDPBS, all embryos were washed twice in modified synthetic oviduct fluid (10 min each). K. R. Bondioli (unpublished observation) found that sheep embryos developed well in synthetic oviduct fluid modified by replacing the 3.2% bovine serum albumin with fetal calf serum. The modification Two or used here employed 10% heat inactivated newborn lamb serum. three embryos were placed in each microdrop of modified synthetic oviduct fluid under paraffin oil at 37 C, in water-saturated 5% CO2 in air atmosphere (15).
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OCTOBER
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T'HERIOGENOLOGY To allow statistical treatment of data by analysis of variance the observed development of the embryos was coded as follows: 0 - no development; 1 - blastocyst; 2 - expanded blastocyst; 3 - hatching blastocyst; 4 - hatched blastocyst. Observations were made every 24 h and the maximum stage reached was recorded during 96 h of culture. This is a modification of a system used to find the cleavage index of bovine embryos in vitro (16). The data were analyzed by the general linear models procedure of the statistical analysis system (17) in the Washington State University computer. A separate trial with 18 embryos collected from seven ewes at a different time was also conducted. Three embryos were loaded into each of six sucrose dilution 0.5-ml French straws. The cryoprotectant was 1.4 M glycerol in MDPBS and the sucrose concentration was 1.0 M in MDPBS. All the straws were fast-frozen as previously described. Thawing and diluting procedures were performed in the same manner as for the fast-frozen 0.25 M sucrose dilution straws. Embryo culture and viability assessment were performed as described by Willadsen (7). RESULTS The number of embryos developed in vitro after each of the four freeze-thaw treatments is given in table 1. The proportion of embryos showing any development (received a score of 1 or more) is also given for each treatment. One embryo was not recovered from one straw in each of the two treatments using standard dilution. It could not be determined if this loss was due to a treatment effect, so these embryos were excluded from the calculations. Eighty percent of the slow-freeze standard dilution and 69% of the fast-freeze standard dilution treatments showed some development in vitro. Both the slow-freeze sucrose dilution and fast-freeze sucrose dilution treatments had only 33% of the embryos showing any development in vitro. TABLE 1.
EFFECT OF FREEZE-THAW AND CRYOPROTECTANT DILUTION PROCEDURES ON THE DEVELOPMENT OF SHEEP MORULAE IN VITRO
Freeze-thaw procedure
Cryoprotectant dilution procedure
Proportion Development scoresa 0 12 3 4
0 deve;oping,
bSlow Slow
Standard 0.25 M Sucrose
3 10
2 4
2 0
4 0
2 1
80 33
bFast Fast
Standard 0.25 M Sucrose
4 10
114 2
1
1
3 1
69 33
aDevelopment was scored at the cessation of culture as: 0 - no development; 1 - blastocyst; 2 - expanded blastocyst; 3 - hatching blastocyst; 4 - hatched blastocyst. b One embryo not recovered after thawing.
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THERIOGENOLOGY Because the interaction between the freeze-thaw procedure and the dilution procedure was not significant, the results were pooled for statistical analysis of the main effects. The average development score for each main treatment are given in table 2. The freeze-thaw procedures were not found to be significantly different. Embryos in the fast freeze-thaw procedure had a mean development score of 1.3 f 0.3 and those in the slow freeze-thaw procedure a mean score of 1.2 + 0.3. There was a highly significant (P 5 0.001) difference in the mean development scores for each dilution procedure. The 0.25 M sucrose dilution was inferior to the standard dilution procedure with development scores of 0.6 + 0.2 and 2.0 f 0.3, respectively.
TABLE
2.
EFFECT OF FREEZE-THAW AND CRYOPROTECTANT ON THE MEAN DEVELOPMENT OF SHEEP MORULAE
Freeze-thaw and Cryoprotectant dilution
Freeze-thaw
PROCEDURES
Development scorea, x + SE
embryos
procedure
Fast
28
1.4 + 0.3
Slow
28
1.2 f 0.3
Standard
26
2.0 * 0.3b
.25 M sucrose
30
0.6 f 0.2'
Cryoprotectant procedure
aFor
Total
DILUTION IN VITRO
explanation
"cValues
with
dilution
of development different
scores
superscripts
see table differ
1.
(PLO.001).
The same mean development for the standard dilution and 0.25 M sucrose dilution procedures are given in table 3, along with the mean development score for the separate 1.0 M sucrose dilution trial. Statistical comparisons of the 1.0 M sucrose procedure to the other two dilution procedures are not relevant because they were conducted on separate groups of embryos. A strictly visual comparison indicates that the 1.0 M sucrose dilution procedure may be marginally superior to the 0.25 M sucrose dilution. However, this 1.0 M sucrose dilution The sucrose is not as effective as the standard dilution procedure. dilution embryos in general had less extruded cellular material than the dilution embrvos immediatelv after completing of the dilution procedure. Post-dilution embryo morphology was optimum with 1.0 M sucrose dilution procedures.
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TABLE
3.
EFFECT OF CRYOPROTECTANT OILUTION PROCEDURES DEVELOPMENT OF FROZEN-THAWED SHEEP MORULAE
Cryoprotectant procedure
dilution Total
ON THE
IN VITRO
Development scorea, x f SE
embryos
Standard
26
2.0 f 0.3
.25 M Sucrose
30
0.6 + 0.2
1.00
M Sucrose
16
1.0 f 0.3
aFor
explanation
One to form varying embryos embryos as shown
of development
scores
1.
embryo in figure 2 appears to be vesiculating, but developed a hatched blastocyst. This vesiculated appearance with amounts of dead and extruded material was commonly seen in Nine out of the 28 that developed after freezing and thawing. (32%) in the fast-thaw treatment had cracked zone pellucidae in figure 2. No cracked zonae were observed in the slow-thaw
A
Figure
see table
2.
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One typical sheep embryo one typical sheep embryo Magnification 100X
1984 VOL. 22 NO. 4
B
with damaged zona pellucida in early development.
and
439
THERIOGENOLOCY procedure. development
Damage to the zona in vitro.
pellucida
did
not affect
embryo
DISCUSSION The fast and slow freeze-thaw methods used with the normal dilution procedures are virtually identical to procedures used in previous studies (4, 5, 7). The percentage of embryos developing in vitro from these procedures agrees with the results reported for frozen-thawed sheep embryos by Willadsen et al. (18) and Willadsen (4) and with a report by Polge and Willadsen (6), which suggests that the fast and slow freeze-thaw methods do not have different abilities to promote the in vitro development of sheep embryos. The 0.25 M sucrose in MDPBS dilution procedure was not found to be effective as a standard procedure in developing sheep embryos in vitro. There was also no significant interaction between the freeze-thaw procedure and the dilution procedure. The 1.0 M sucrose in MDPBS dilution procedure was not as effective as the standard dilution procedure. Due to the limited number of embryos employed, further experimentation with 1.0 M sucrose will be necessary. A. 0. Trounson (personal communication) has found sucrose concentrations in the range of 1.0 M to be effective for cryoprotectant dilution of sheep embryos. A number of cracked zona pellucidae were noted in the fast frozen-thawed embryos. Damage to the zona pellucida has not been shown to affect the viability of frozen-thawed embryos (7, 19). However, it does make the embryos more sensitive to disruption during manipulation. S. P. Leibo (personal communication) has indicated that thawing rates slower than those produced by a 37 C waterbath thaw will reduce zone damage. Kasai et al. (13) demonstrated that mouse embryos thawed at 360 C/min (37 C water bath) were more viable in vitro than embryos thawed at 25 C/min (room-temperature air thaw). Further study will be required to establish the optimum warming rate and the methods needed to reproduce it consistently in the field. After 96 h in culture, all the frozen-thawed embryos appeared to be dead or dying. Many embryos had numerous vesicles and extruded dead material. The embryos that appeared to be vesiculating were generally the only embryos to develop to at least the blastocyst stage. Many cells are killed by the freeze-thaw or dilution process, but the surviving cells may reform a blastocyst. Dead material is extruded from the living cell mass and can be seen as dark patches on the exterior of most blastocysts. The morphological appearance of embryos from the 0.25 M sucrose dilution was superior to that of embryos treated by the standard dilution procedure. The appearance of the 1.0 M sucrose-diluted embryos was superior to that of the 0.25 M sucrose-diluted embryos. The morphological evaluation of these embryos after cryoprotectant dilution does not correlate well with their ability to develop in vitro. Elsden et al. (20) noted that post-thaw morphological evaluation does not correlate well with in vivo development of frozen-thawed bovine embryos.
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THERIOGENOLOGY The one-step dilution system with a sucrose gradient appears to be a simple and efficient method to thaw embryos and is being used successfully with bovine embryos. However, this study suggests that ovine embryos have a more reduced viability in culture following sucrose dilution when compared with standard dilution techniques.
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THERIOGENOLOGY REFERENCES 1.
Whittingham, D.G. Survival of mouse embryos after freezing and thawing. Nature 233:125-126. (1971)
2.
Whittinghan, D.G., Leibo, S.P. and Mazur, P. Survival of mouse embryos frozen to -196 and -296 C. Science 178:411-414. (1972)
3.
Wilmut, I. and Rowson, L.E.A. The successful low-temperature preservation of mouse and cow embryos. J. Reprod. Fertil. a:252 (Abstr.). (1973)
4.
Willadsen, S.M. Deep freezing of sheep embryos. In: Elliott, K. and Whelan, J. (Eds) The Freezing of Mammalian Embryos, Ciba Fndn. Symp. No. 52 (new series). Elsevier/North Holland, Amsterdam, 1977, pp. 175-189.
5.
Willadsen, S.M., Polge, C. and Rowson, L.E. The viability of deep-frozen cow embryos. J. Reprod. Fertil. 52:391-393. (1978)
6.
Polge, C. and Willadesn, S.M. Freezing eggs and embryos of farm animals. Cryobiology 15:370-373. (1978)
7.
Willadsen, S.M., Polge, C. and Rowson, L.E. In vitro storage of cattle embryos. In: Control of Reproduction In the Cow. Commission of theTuropean Communities, The Hague, Netherlands, 1978, pp. 427-436.
8.
Whittingham, D.G., Wood, M., Farrant, J., Lee, H. and Halsey, J.A. Survival of frozen mouse embryos after rapid thawing from -196 C. J. Reprod. Fertil. !&:ll-21.
9.
Lehn-Jensen, H. bovine embryos.
Bovine egg transplantation. Preservation of Nord. Vet. Med. 2:523-532. (1980)
10.
Renard, J.-P., Heyman, Y. and Ozil, 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. Med. Vet. 126:23-32. (1982)
11.
Leibo, S.P. Field trial of one-step frozen bovine embryos transferred non-surgically. Theriogenology 19:139 (Abstr.). (1984)
12.
Renard, J.-P., Heyman, Y., Leymonie, P. and Plat, J.-C. Sucrose dilution: A technique for field transfer of bovine embryos frozen in the straw. Theriogenology -19:145 (Abstr.). (1983)
13.
Kasai, M., Niwa, K. and Iritani, A. Survival of mouse embryos frozen and thawed rapidly. J. Reprod. Fertil. 2:51-56. (1980)
14.
Wright, R.W., Jr., Bondioli, K., Grammer, J., Kuzan, F.and Menino, Jr., A. FSH or LH plus FSH superovulation in ewes following estrus synchronization with medroxprogesterone acetate pessaries. J. Anim. Sci. 52:115-118. (1981)
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THERIOGENOLOGY
15.
Brinster, R.L. A method for in vitro cultivation of mouse ova from two-cell to blastocyst. Exp. Cell. Res. 32:205-208. (1963)
16.
Wright, R.W., Jr., Anderson, G.B., Cupps. P.T. and Orost, M. Successful culture in vitro of bovine embryos to the blastocyst stage. Biol. Reprod. -14:157-162. (1976)
17.
Barr, J.J., Goodnight, J.H., Sall, J.P., Blair, W.H., Chilka, D.M., Council, K.A. and Helwig, J.T. SAS User's Guide, SAS Institute, Inc., Raleigh, NC, 1979, pp.
18.
Willadsen, S.M., Polge, C., Rowson, L.E. and Moor, R.M. Deep 46:151-154. (1976) freezing of sheep embryos. J. Reprod. Fertil. -
19.
Massip, A., Van der Zwalmen, P., Factors, F., DeCoster, R., D'Ieteren, G. and Hanzen, C. Deep freezing of cattle embryos in glass ampules of french straws. Theriogenology 12:79-84. (1979)
20.
Elsden, R.P., Seidel, Jr., G. E., Takeda, T. and Farrand, G.D. Field experiments with frozen-thawed bovine embryos transferred nonsurgically. Theriogenology 17:1-10. (1982)
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