- Email: [email protected]
thawed bovine demi-embryos
THERIOGENOLOGY
IN VITRO SURVIVAL OF FRESH AND FROZEN/THAWED BOVINE DEMI-EMBRYOS A. Lucas-Hahn and H. Niemann Institut ftir Tierzucht und Tierverhalten (FAL), Mariensee 3057 Neustadt 1, Germany Received for publication: April! 24, 1991 Accepted: &,dy 22, 1991 ABSTRACT Three experiments were conducted to investigate the effects of type of culture medium in freshly bisected bovine embryos and the effects of agar embedding and of 1.2 propanediol (PROH) as the cryoprotectant in frozen/thawed bisected bovine embryos on development in vitro. A total of 265 bovine embryos were used as controls or were microsurgically bisected and were cultured in vitro for 48 hours and development was determined 24 and 48 hours after the onset of culture. Whitten’s medium supported more (P < 0.05) intact and demi-embryos to grow to expanded blastocysts (92.9 and 73.1%, res ectively) compared with Ham’s F10 (43.8 and 26.3%, respectively) and PBS P53.8 and 12.5%? respectively). Embedding in agar and culture in Whitten’s medium resulted in a higher (P < 0.05) percentage of m vitro development of frozen/thawed demiembryos after 24 hours than the freezing of nonembedded demi-embryos (44.1 versus 19.6%, respectively). This difference disap eared, however, after a 48 hours culture period (17.6 versus 11.8%, respectively P. Following freezing in PROH, survival rates of 40 and 28%, respectively after 24 hours of culture were obtained for intact and den&embryos. The respective percentages after 48 hours were 8.6 and 16%. Since neither embedding in agar nor the use of PROH as the cryoprotectant resulted in high survival rates of frozen/thawed den&embryos in vitro, new freezing procedures are needed to overcome the sensitivity of demiembryos to freezing and thawing. Key words: bovine, embryo, freezing, bisection, in vitro INTRODUCTION Lehn-Jensen and Willadsen (1) were the first to produce liveborn calves from frozen/thawed den-ii-embryos. Their method was rather complicated and involved the splitting of early morulae followed by a temporary in vivo culture in ligated sheep oviducts prior to the freezing of the agar embedded demi-blastocysts.
Acknowledgments: This project was supported by the H. Wilhelm Schaumann Stiftung, Hamburg, Germany. The authors thank Mr. H.-H. Dijpke and Mr. K.-G. Hadeler for their skilled technical assistance.
OCTOBER 1991 VOL. 36 NO. 4
619
THERIOGENOLOGY
Nowadays, the production of viable den&embryos can be accomplished with rather simple methods (2-4). However the freezing of demi-embryos results in lower survival rates compared with that of intact embryos (5-8). It has been proposed that the sensitivrty of den&embryos to the freezing and thawing process is mainly dependent on the punctured zona pellucida (9) and/or the reduced number of cells (5). To improve the survival rates of frozen/thawed den&embryos it was suggested that the punctured zona pellucida be sealed by an additional zona ellucida (9) or that the den&embryos be embedded in agarchips (7,8) or the Preezing procedure be modified (5). The purpose of our study was to evaluate the developmental capacity of bovine denn-embryos in the in vitro culture before and after cryopreservation. In the first experiment, the in vitro development of bovine den&embryos was compared in three different media, In the second experiment, the effects of sealing the zona pellucida fracture with an agarchip were investigated. In the third experiment a freezing procedure used successfully on early stage embryos with PROH as the cryoprotectant, was applied to bovine den&embryos. MATERIALS
AND METHODS
During the period from May 1987 to August 1989 a total of 114 Holstein Friesian dairy cows were superovulated using either 2500 IU PMSG (Intergonan, Vemie, Germany) or 40 mg FSH (FSH-P, Burns Biotec) on Days 9 to 13 of the estrous cycle. The animals were inseminated with frozen/thawed semen 48 and 60 hours after superovulation. Morulae and blastocysts were collected nonsur ‘tally on Day 7 after AI by flushing the uterine horns with Dulbecco’s PBS (E PBS, Sigma, St. Louis, MO, U.S.A.) supplemented with 1% newborn calf serum (NBCS, Boehringer, Mannheim, Germany). Embryos were evaluated according to morphological criteria (10); only embryos classified as excellent or good were used for the experiments. A total of 265 morphologically intact, late morulae and blastocysts were used either as controls (intact embryos; n = 156) or after microsurgical bisection (n = 109) to give rise to 198 morphologically intact den&embryos. Embryo bisection was carried out at room temperature using two Leitz micromanipulators and a Wild stereomicroscope at a 100x magnification. The embryos were fixed by negative pressure using a holding pipette (outer diameter 120 pm). In blastocysts, the inner cell mass was oriented towards the microsurgical blade so that the embryo could be bisected symmetrically. Approximately half of the diameter of the zona pellucida and a part of the embryo were cut by a descending movement of the microscalpel (Spezialklinge “s”, Klein, Heidelberg, Germany). Bisection was completed by cutting the intact part of the embryo through a second up and down movement of the mcroblade. One demi-embryo was transferred into the original zona pellucida with the aid of the microblade. The other demi-embryo was reinserted into an evacuated zona pellucida, ori ’ ated from degenerated embryos or from unfertilized ova which had been store CYm the freezer at -18°C until use. Subsequently all demi-embryos were either cultured in vitro or after a short-term culture of 1 to 3 hours subjected to cryopreservation. Demi and intact embryos were cultured in 35-mm petri dishes at 37°C in a humidified atmosnhere at 5% CO? in air. Moroholoaical develoDment to expanded, hatching or hatchedblasto&ts was evaluatkd 24&d 48 hour; after the onset of culture. Frozen/thawed embryos were additionally controlled at 4 hours after the onset of culture. In Experiment 1, Whitten’s, Ham’s FlO and PBSmedium each supplemented with 20% NBCS were compared in their efficiency to 620
OCTOBER 1991 VOL. 36 NO. 4
THERIOGENOLOGY
support embryo development. In Experiments 2 and 3 the embryos were cultured in Whitten’s medium supplemented with 20% NBCS. In Experiment 2, demi-emb OS were embedded in agar as described by Willadsen (11) to seal the zonae pe‘1’lucidae. Then, intact and den&embryos were equilibrated in a 1.4-M glycerol solution (Merck, Darmstadt, Germany) at room temperature for 20 minutes before they were loaded individually or as monozygotic pairs into 0.25ml straws. After sealing the open ends with a marker stick, the straws were transferred to an ethanol bath (Ultratemp 2000, FCSOHC, Julabo, Freiburg, Germany) retooled to -7°C. Crystallization was induced with a supercooled forceps at -7°C Pollowed by a holding period of 5 minutes. The straws were cooled from -7°C to -28°C at 03”C/min and from -28°C to -35°C at O.l”C/min before being plunged into liquid nitrogen (12). In Experiment 3, intact and demi-embryos were equilibrated at room temperature m a 1.5-M PROH solution (Riedel de Haen, Seelze, Germany) for 15 minutes followed by a second step in 1.5 M of PROH plus 0.2 M of sucrose (Serva, Heidelberg, Germany) for 5 minutes before they were loaded into 0.25~ml straws that were sealed with a marker stick. The straws were then transferred to the precooled ethanol bath (-7°C) and seeding was induced with a supercooled forceps. After a 5-minute holding period, the samples were cooled from 7°C to 30°C at 0.3”C/minute. After a holding period of 15 minutes at -3O”C, the straws were plunged into liquid nitrogen. Samples in Experiments 2 and 3 were thawed in air at room temperature. The cryoprotectants were removed using sucrose in two steps. In the first step, the embryos wee transferred into a solution of 0.7 M of glycerol or 0.75 M of PROH, respectively, each supplemented with 0.7 M of sucrose for 5 to 10 minutes. As the second step, the embryos were pipetted into a 0.7-M sucrose solution (5 to 10 minutes). Subsequently, the embryos were washed three times in PBS supplemented with 20% NBCS before they were evaluated according to morphological criteria (10) and were then cultured in vitro. Embryos classified as excellent, good or fair after thawin were considered to have survived. The data were analyzed by Chi-square, and dp. rfferences were considered significant at P < 0.05. RESULTS In Experiment 1, Whitten’s medium was shown to support development of both intact and den&embryos significantly (P < 0.05) better than the two other media (Table 1). After 48 hours in vitro culture the development of intact embryos in Ham’s FlO and PBS medium was higher (P < 0.05) than that of the demiembryos. In Experiment 2, the survival rate of frozen/thawed agar embedded demiembryos after 24 hours of culture was higher (P < 0.05) than that of the nonembedded demi-embryos (Table 2). Overall, developmental rates were lower than in Experiment 1. In Experiment 3, similar survival rates were found between demi and intact embryos using PROH as the cryoprotectant (Table 3). Abnormal pressure conditions were found in 34% of the straws. Some straws exploded, which led to the loss of 9.8% of the control and 14.6% of the demi-embryos.
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THERIOGENOLOGY
Table 1.
Developmental capacity in vitro of demi and intact embryos in three dtfferent media
Medium
Intact embryos
Whitten’s Ham’s FlO PBS
Demiembryos
Whitten’s Ham’s FlO PBS
No. of embryos
:: 13
No. developing after 24 hours in vitro culture n (W
No. developing after 48 hours in vitro culture n (W
13 9 9
26 ::
a - e: Means in the same column without a common superscript differ significantly (P < 0.05).
DISCUSSION The results of Experiment 1 indicate that Whitten’s medium a simple bicarbonate buffered medium, was superior to Ham’s FlO, a complete bicarbonate buffered medium, and PBS, a simple phosphate buffered medium, in the support of intact and bisected bovine embryo development. Similar results concerning simple and complete media were reported for mtact mouse and hamster embryos (13,14). Den&embryos seemed to react with more sensitivity to the different types of culture media than intact embryos. Whereas in intact embryos significant differences among the media were found after a 48-hours culture period, the development of demi-embryos already differed significantly after 24 hours of in vitro culture. These differences remained after an additional 24 hours of culture. Our results were in agreement with the findings from other investigators (15) who observed a low developmental rate of den&embryos in PBS and in Ham’s FlO after 48 hours of culture. It has been shown that an intact zona pellucida is not essential for the survival of Day 7 embryos after freezin and thawing (16,17). However, a different role for the zona pellucida during the l!reezing of den&embryos was postulated. It was assumed that in den&embryos the sealing of the zona pellucida fracture can prevent the loss of additional blastomeres simply by having better physical protection against the growth of ice crystals (16). Several studies suggested that survival rates of den&embryos could be improved after sealin the zona pellucida with an agarchip (7,18) or with an additional zona pellucida (9! . Similarly with the observation of other investigators (17) our study indicates that the presence of additional protection by the agar does not result in better survival of demiembryos after freezing and thawing. A further repairing of the zona pellucida fracture therefore seems not to be necessary; however, this has to be substantiated by further study of in vivo development.
622
OCTOBER 1991 VOL. 36 NO. 4
Z P
k
f ?
iii
57
39
Den&embryos
Den&embryos embedded in agar 34 (97.1)
51 (91.1)
56 (98.2) 35 (89.7)
60 (100)
(%)
(W 60 (96.8)
after thawing
Recovery rate after thawing
(79.4)
(76.5)
39/51 27/34
(75)
45/60
Viable after 4 hours of culture (%)
ab: Means in the same column without a common superscript differ significantly (P < 0.05).
62
No. of embryos frozen
(44.l)b
(19.6)a
lo/51 15/34
(33.3)ab
20/60
embrvos after 24 hours of culture (W
In vitro development of frozen-thawed den&embryos, with or without embedding in agar
Intact embryos (controls)
Table 2.
6/34 (17.6)
6/51 (11.8)
lo/60 (16.7)
after 48 hours of culture (%)
51
41
Den&embryos
No. of frozen embryos
(90.2) (85.4)
35
(76.1) (71.4)
35 25
(W
(W
46
after thawing
Recovery rate after thawing
15/25
(60)
(74.3)
Viable after 4 hours of culture (W
26/35
In vitro development of den&embryos after freezing in 1,2 propanediol
Intact embryos
Table 3.
7/25
14/35
(28)
(40)
embrvos after 24 hours of culture (So)
.4/25 (16)
3/35 (8.6)
after 48 hours of culture (S/o>
THERIOGENOLOGY
The survival rates of frozen-thawed den&embryos varied between 36 and 77% and the developmental rates between 0 and 33% (7-9,17-20). We obtained high survival rates immediately after thawing, and the den&embryos retained their viability during the first 24 hours of in vitro culture. This was probably the result of the short-term culture of the demi-embryos after splitting and before freezing. During this period reorganization of the den&embryos could occur., and damaged blastomeres were extruded and the remaining cells formed a new mtact cell unit. Previous- investigations (8,21) have shown a beneficial effect of a short-term culture after sphtting and prior to freezing of the demi-embryos. The reason for the low developmental rates after 48 hours of in vitro culture is not known. As shown in our first experiment, Whitten’s medium supported development of fresh ossible that frozen-thawed intact and demi-embryos equally well. It is therefore embryos have different culture requirements than fres ! embryos. This needs to be investigated in future studies. Some investigators (5) have suggested new freezing procedures for demiembryos. Attempts to improve the survrval rates of den&embryos by vitrification have failed (8,20)_ Our third experiment was based on the assumption that demiembryos behave like early embryonic stages (2, 8 and 16 cells) during the cryo reservation process due to their reduced number of cells. PROH was used for oprotectant to freeze bovine den&embryos but turned out the L st time as the or bisected embryos. PROH was shown to be more effective to be not appropriate Yfy than glycerol or DMSO in the freezing of very early embryonic stages in the mouse, rabbit and human (22-24). A potential explanation for the abnormal pressure conditions found in Experiment 3 could have been the use of marker however, worked well in sticks for sealing the straws. This method, cryopreservation wrth lycerol as the cryoprotectant, and the batches of straws and suckers were identic 3 The abnormal pressure conditions were likely caused b interactions between PROH and liquid nitrogen. The marker sticks probably le fvt the ends at the straws leaky through which liquid nitrogen could enter and cause high ressure upon thawing. Abnormal high pressure inside the straws was not in recent experiments (unpublished foun B when the straws were heat-sealed observation). This observation shows that sealing requirements can differ in relation to the cryoprotectant used. The abnormal pressure conditions which sometimes lead to movement of the different segments inside the straws can at least partially explain the lower survival rates found in Experiment 3 compared with those found in Experiment 2. In conclusion, our study indicates that more experiments are needed to overcome the enormous sensitivity of bovine demiembryos to cryopreservation, with the goal of obtaining similarly high survival rates as those for intact embryos.
REFERENCES L&n-Jensen, H. and Willadsen S. M. Deep-freezing embryos. Theriogenology B:427-432 (1983).
of cow half and quarter
Williams,.T.J., Elsden, R. P. and Seidel Jr., G. E. Identical twin bovine pregnancres derived from bisected embryos. Theriogenology 12: 114 abstr. (1982). Williams, T. J., Elsden, R. P. and Seidel Jr., G. E. Pregnan bisected bovine embryos. Theriogenology 22:521-531(1988
OCTOBER 1991 VOL. 36 NO. 4
rates with
4.
Massip, A, Van der Zwalmen, A. and Ectors, F. Production de jumeaux monozygotes par duplication d’embryons chez les bovins. Ann. Med. Vet. 129:53-57 (1985).
5. Heyman Y. and Chesne, P. Freezing bovine embryos: survival after transfer of one half, one or two blastocysts frozen in straws.Theriogenology U:241 abstr. (1984) 6. Picard, L., King, W. A. and Betteridge, K. J. Production of sexed calves from frozen thawed embryos. Vet. Rec. m:603-608 (1985). 7. Rorie,R. W., Pendleton, R. J., Youngs, C. R. and R. A. Godke. Viability of den&embryos produced before vs. after deep freezing. Theriogenology a:192 abstr. (1986). 8. Massip, A., Van der Zwalmen, P. and Ectors F. Cryopreservation of bovine half embryos by conventional and vitrification methods. In: T. Greve (ed.), Proc. of Sym osium: Application of Egg and Embryo Technologies to Domestic Animals QRoyal Veterinary and Agricultural University, Copenhagen) (1987). 9. Niemann, H., Brem, G., Sacher, B., Smidt, D. and Krausslich, H. An approach to successful freezing of demi-embryos derived &om Day 7 bovine embryos. Theriogenology 25:519-524 (1986). 10. Niemann, H. Miiglichkeiten und Grenzen des Embryotransfers bei landwirtschaftlichen Nutztieren. Tierarztl. Umsch. &&625-633 (1986). 11. Willadsen, S. M. A method for culture of micromanipulated sheep embryos and its use to produce monozygotic twins. Nature m:298-300 (1979). 12. Niemann, H. Freezing bovine embryos: effects of a one-step addition of 1.4 M glycerol. Theriogenology 23:369-379 (1985). 13. Biery K. A. and Kraemer D. C. Comparison of Ham’s FlO, modified Whitten’s and modified Eagle’s medta supplemented with either bovine serum albumin or newborn calf serum for the in vitro culture of 8-cell mouse embryos. Theriogenology a:225 abstr. (1988). 14. Bavister, B. D. Studies on developmental blocks in cultured hamster embryos. In: B.D. Bavister (ed.), The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation In Vitro, Plenum Press, New York, 1987, pp. 219-249. 15. Wurth, Y. A. and Kruip, T. A. M. Evaluation of different media for culture of bovme demi embryos. 11th Int. Cong. Anim. Reprod. and AI (Dublm, Ireland) 1. : 484 abstr. (1988). 16. Niemann, H., Pryor, J. H. and Bondioli, K. R. Effects of slitting the zona pellucida and its subsequent sealing on freeze-thaw survival of Day 7 bovine embryos.Theriogenology a:675681 (1987). 17. Picard, L., Schneider, U., Betteridge, K. J. and King, W.A. Effects of the zona pellucida, agar-embedding and culture on the survival of micromanipulated bovine embryos after freezing and thawing. J. In Vitro Fertil. Emb. Transf.5:268-274 (1988). OCTOBER 1991 VOL. 36 NO. 4
18. Tsunoda, Y., Tokmagy, T., Okubo, Y. and Sugie, T. Beneficial effect of agar for the frozen storage of bisected embryos. Theriogenology 28: 317-322 (1987). 19. Takeda, T., Henderson, B. and Hasler J.F. Dee freezing of split and intact bovine embryos. Theriogenology a:285 abstr. Q1987). 20. Bielanski, A. and Hare, W.C.D. Survival in vitro of bovine embryos after freezing by slow cooling rates or vitrification. Theriogenology a:223 abstr. (1988). 21. Chesne, P., Heyman, Y., Chupin, D., Procureur, R. and Menezo, Y. Freezing cattle den&embryos: influence of a eriod of culture between splitting and freezing on survival. Theriogeno Pogy a:218 abstr. (1987). 22. Renard, J.P., Bui-Xuan-Nguyen and Gamier, V. Two-step freezing of twocell rabbit embryos after partial dehydration at room temperature. J. Reprod. Fertil. Z:573-580 (1984). 23. Hemandez-Ledezma, J. J. and Wright, R.W. Jr., Deep-freezing of mouse one-cell embryos and oocytes using different cryoprotectants. Theriogenology Z:735-743 (1989). 24. Siebzehnruebl, E. R., Todorow, S., Van Uem, J., Koch, R., Wildt, L. and Lang, N. Cryopreservation of human and rabbit oocytes and one-cell embryos: a corn arison of DMSO and propanediol. Hum. Reprod. &312-317 (1989 P.
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IN VITRO SURVIVAL OF FRESH AND FROZEN/THAWED BOVINE DEMI-EMBRYOS A. Lucas-Hahn and H. Niemann Institut ftir Tierzucht und Tierverhalten (FAL), Mariensee 3057 Neustadt 1, Germany Received for publication: April! 24, 1991 Accepted: &,dy 22, 1991 ABSTRACT Three experiments were conducted to investigate the effects of type of culture medium in freshly bisected bovine embryos and the effects of agar embedding and of 1.2 propanediol (PROH) as the cryoprotectant in frozen/thawed bisected bovine embryos on development in vitro. A total of 265 bovine embryos were used as controls or were microsurgically bisected and were cultured in vitro for 48 hours and development was determined 24 and 48 hours after the onset of culture. Whitten’s medium supported more (P < 0.05) intact and demi-embryos to grow to expanded blastocysts (92.9 and 73.1%, res ectively) compared with Ham’s F10 (43.8 and 26.3%, respectively) and PBS P53.8 and 12.5%? respectively). Embedding in agar and culture in Whitten’s medium resulted in a higher (P < 0.05) percentage of m vitro development of frozen/thawed demiembryos after 24 hours than the freezing of nonembedded demi-embryos (44.1 versus 19.6%, respectively). This difference disap eared, however, after a 48 hours culture period (17.6 versus 11.8%, respectively P. Following freezing in PROH, survival rates of 40 and 28%, respectively after 24 hours of culture were obtained for intact and den&embryos. The respective percentages after 48 hours were 8.6 and 16%. Since neither embedding in agar nor the use of PROH as the cryoprotectant resulted in high survival rates of frozen/thawed den&embryos in vitro, new freezing procedures are needed to overcome the sensitivity of demiembryos to freezing and thawing. Key words: bovine, embryo, freezing, bisection, in vitro INTRODUCTION Lehn-Jensen and Willadsen (1) were the first to produce liveborn calves from frozen/thawed den-ii-embryos. Their method was rather complicated and involved the splitting of early morulae followed by a temporary in vivo culture in ligated sheep oviducts prior to the freezing of the agar embedded demi-blastocysts.
Acknowledgments: This project was supported by the H. Wilhelm Schaumann Stiftung, Hamburg, Germany. The authors thank Mr. H.-H. Dijpke and Mr. K.-G. Hadeler for their skilled technical assistance.
OCTOBER 1991 VOL. 36 NO. 4
619
THERIOGENOLOGY
Nowadays, the production of viable den&embryos can be accomplished with rather simple methods (2-4). However the freezing of demi-embryos results in lower survival rates compared with that of intact embryos (5-8). It has been proposed that the sensitivrty of den&embryos to the freezing and thawing process is mainly dependent on the punctured zona pellucida (9) and/or the reduced number of cells (5). To improve the survival rates of frozen/thawed den&embryos it was suggested that the punctured zona pellucida be sealed by an additional zona ellucida (9) or that the den&embryos be embedded in agarchips (7,8) or the Preezing procedure be modified (5). The purpose of our study was to evaluate the developmental capacity of bovine denn-embryos in the in vitro culture before and after cryopreservation. In the first experiment, the in vitro development of bovine den&embryos was compared in three different media, In the second experiment, the effects of sealing the zona pellucida fracture with an agarchip were investigated. In the third experiment a freezing procedure used successfully on early stage embryos with PROH as the cryoprotectant, was applied to bovine den&embryos. MATERIALS
AND METHODS
During the period from May 1987 to August 1989 a total of 114 Holstein Friesian dairy cows were superovulated using either 2500 IU PMSG (Intergonan, Vemie, Germany) or 40 mg FSH (FSH-P, Burns Biotec) on Days 9 to 13 of the estrous cycle. The animals were inseminated with frozen/thawed semen 48 and 60 hours after superovulation. Morulae and blastocysts were collected nonsur ‘tally on Day 7 after AI by flushing the uterine horns with Dulbecco’s PBS (E PBS, Sigma, St. Louis, MO, U.S.A.) supplemented with 1% newborn calf serum (NBCS, Boehringer, Mannheim, Germany). Embryos were evaluated according to morphological criteria (10); only embryos classified as excellent or good were used for the experiments. A total of 265 morphologically intact, late morulae and blastocysts were used either as controls (intact embryos; n = 156) or after microsurgical bisection (n = 109) to give rise to 198 morphologically intact den&embryos. Embryo bisection was carried out at room temperature using two Leitz micromanipulators and a Wild stereomicroscope at a 100x magnification. The embryos were fixed by negative pressure using a holding pipette (outer diameter 120 pm). In blastocysts, the inner cell mass was oriented towards the microsurgical blade so that the embryo could be bisected symmetrically. Approximately half of the diameter of the zona pellucida and a part of the embryo were cut by a descending movement of the microscalpel (Spezialklinge “s”, Klein, Heidelberg, Germany). Bisection was completed by cutting the intact part of the embryo through a second up and down movement of the mcroblade. One demi-embryo was transferred into the original zona pellucida with the aid of the microblade. The other demi-embryo was reinserted into an evacuated zona pellucida, ori ’ ated from degenerated embryos or from unfertilized ova which had been store CYm the freezer at -18°C until use. Subsequently all demi-embryos were either cultured in vitro or after a short-term culture of 1 to 3 hours subjected to cryopreservation. Demi and intact embryos were cultured in 35-mm petri dishes at 37°C in a humidified atmosnhere at 5% CO? in air. Moroholoaical develoDment to expanded, hatching or hatchedblasto&ts was evaluatkd 24&d 48 hour; after the onset of culture. Frozen/thawed embryos were additionally controlled at 4 hours after the onset of culture. In Experiment 1, Whitten’s, Ham’s FlO and PBSmedium each supplemented with 20% NBCS were compared in their efficiency to 620
OCTOBER 1991 VOL. 36 NO. 4
THERIOGENOLOGY
support embryo development. In Experiments 2 and 3 the embryos were cultured in Whitten’s medium supplemented with 20% NBCS. In Experiment 2, demi-emb OS were embedded in agar as described by Willadsen (11) to seal the zonae pe‘1’lucidae. Then, intact and den&embryos were equilibrated in a 1.4-M glycerol solution (Merck, Darmstadt, Germany) at room temperature for 20 minutes before they were loaded individually or as monozygotic pairs into 0.25ml straws. After sealing the open ends with a marker stick, the straws were transferred to an ethanol bath (Ultratemp 2000, FCSOHC, Julabo, Freiburg, Germany) retooled to -7°C. Crystallization was induced with a supercooled forceps at -7°C Pollowed by a holding period of 5 minutes. The straws were cooled from -7°C to -28°C at 03”C/min and from -28°C to -35°C at O.l”C/min before being plunged into liquid nitrogen (12). In Experiment 3, intact and demi-embryos were equilibrated at room temperature m a 1.5-M PROH solution (Riedel de Haen, Seelze, Germany) for 15 minutes followed by a second step in 1.5 M of PROH plus 0.2 M of sucrose (Serva, Heidelberg, Germany) for 5 minutes before they were loaded into 0.25~ml straws that were sealed with a marker stick. The straws were then transferred to the precooled ethanol bath (-7°C) and seeding was induced with a supercooled forceps. After a 5-minute holding period, the samples were cooled from 7°C to 30°C at 0.3”C/minute. After a holding period of 15 minutes at -3O”C, the straws were plunged into liquid nitrogen. Samples in Experiments 2 and 3 were thawed in air at room temperature. The cryoprotectants were removed using sucrose in two steps. In the first step, the embryos wee transferred into a solution of 0.7 M of glycerol or 0.75 M of PROH, respectively, each supplemented with 0.7 M of sucrose for 5 to 10 minutes. As the second step, the embryos were pipetted into a 0.7-M sucrose solution (5 to 10 minutes). Subsequently, the embryos were washed three times in PBS supplemented with 20% NBCS before they were evaluated according to morphological criteria (10) and were then cultured in vitro. Embryos classified as excellent, good or fair after thawin were considered to have survived. The data were analyzed by Chi-square, and dp. rfferences were considered significant at P < 0.05. RESULTS In Experiment 1, Whitten’s medium was shown to support development of both intact and den&embryos significantly (P < 0.05) better than the two other media (Table 1). After 48 hours in vitro culture the development of intact embryos in Ham’s FlO and PBS medium was higher (P < 0.05) than that of the demiembryos. In Experiment 2, the survival rate of frozen/thawed agar embedded demiembryos after 24 hours of culture was higher (P < 0.05) than that of the nonembedded demi-embryos (Table 2). Overall, developmental rates were lower than in Experiment 1. In Experiment 3, similar survival rates were found between demi and intact embryos using PROH as the cryoprotectant (Table 3). Abnormal pressure conditions were found in 34% of the straws. Some straws exploded, which led to the loss of 9.8% of the control and 14.6% of the demi-embryos.
OCTOBER 1991 VOL. 36 NO. 4
621
THERIOGENOLOGY
Table 1.
Developmental capacity in vitro of demi and intact embryos in three dtfferent media
Medium
Intact embryos
Whitten’s Ham’s FlO PBS
Demiembryos
Whitten’s Ham’s FlO PBS
No. of embryos
:: 13
No. developing after 24 hours in vitro culture n (W
No. developing after 48 hours in vitro culture n (W
13 9 9
26 ::
a - e: Means in the same column without a common superscript differ significantly (P < 0.05).
DISCUSSION The results of Experiment 1 indicate that Whitten’s medium a simple bicarbonate buffered medium, was superior to Ham’s FlO, a complete bicarbonate buffered medium, and PBS, a simple phosphate buffered medium, in the support of intact and bisected bovine embryo development. Similar results concerning simple and complete media were reported for mtact mouse and hamster embryos (13,14). Den&embryos seemed to react with more sensitivity to the different types of culture media than intact embryos. Whereas in intact embryos significant differences among the media were found after a 48-hours culture period, the development of demi-embryos already differed significantly after 24 hours of in vitro culture. These differences remained after an additional 24 hours of culture. Our results were in agreement with the findings from other investigators (15) who observed a low developmental rate of den&embryos in PBS and in Ham’s FlO after 48 hours of culture. It has been shown that an intact zona pellucida is not essential for the survival of Day 7 embryos after freezin and thawing (16,17). However, a different role for the zona pellucida during the l!reezing of den&embryos was postulated. It was assumed that in den&embryos the sealing of the zona pellucida fracture can prevent the loss of additional blastomeres simply by having better physical protection against the growth of ice crystals (16). Several studies suggested that survival rates of den&embryos could be improved after sealin the zona pellucida with an agarchip (7,18) or with an additional zona pellucida (9! . Similarly with the observation of other investigators (17) our study indicates that the presence of additional protection by the agar does not result in better survival of demiembryos after freezing and thawing. A further repairing of the zona pellucida fracture therefore seems not to be necessary; however, this has to be substantiated by further study of in vivo development.
622
OCTOBER 1991 VOL. 36 NO. 4
Z P
k
f ?
iii
57
39
Den&embryos
Den&embryos embedded in agar 34 (97.1)
51 (91.1)
56 (98.2) 35 (89.7)
60 (100)
(%)
(W 60 (96.8)
after thawing
Recovery rate after thawing
(79.4)
(76.5)
39/51 27/34
(75)
45/60
Viable after 4 hours of culture (%)
ab: Means in the same column without a common superscript differ significantly (P < 0.05).
62
No. of embryos frozen
(44.l)b
(19.6)a
lo/51 15/34
(33.3)ab
20/60
embrvos after 24 hours of culture (W
In vitro development of frozen-thawed den&embryos, with or without embedding in agar
Intact embryos (controls)
Table 2.
6/34 (17.6)
6/51 (11.8)
lo/60 (16.7)
after 48 hours of culture (%)
51
41
Den&embryos
No. of frozen embryos
(90.2) (85.4)
35
(76.1) (71.4)
35 25
(W
(W
46
after thawing
Recovery rate after thawing
15/25
(60)
(74.3)
Viable after 4 hours of culture (W
26/35
In vitro development of den&embryos after freezing in 1,2 propanediol
Intact embryos
Table 3.
7/25
14/35
(28)
(40)
embrvos after 24 hours of culture (So)
.4/25 (16)
3/35 (8.6)
after 48 hours of culture (S/o>
THERIOGENOLOGY
The survival rates of frozen-thawed den&embryos varied between 36 and 77% and the developmental rates between 0 and 33% (7-9,17-20). We obtained high survival rates immediately after thawing, and the den&embryos retained their viability during the first 24 hours of in vitro culture. This was probably the result of the short-term culture of the demi-embryos after splitting and before freezing. During this period reorganization of the den&embryos could occur., and damaged blastomeres were extruded and the remaining cells formed a new mtact cell unit. Previous- investigations (8,21) have shown a beneficial effect of a short-term culture after sphtting and prior to freezing of the demi-embryos. The reason for the low developmental rates after 48 hours of in vitro culture is not known. As shown in our first experiment, Whitten’s medium supported development of fresh ossible that frozen-thawed intact and demi-embryos equally well. It is therefore embryos have different culture requirements than fres ! embryos. This needs to be investigated in future studies. Some investigators (5) have suggested new freezing procedures for demiembryos. Attempts to improve the survrval rates of den&embryos by vitrification have failed (8,20)_ Our third experiment was based on the assumption that demiembryos behave like early embryonic stages (2, 8 and 16 cells) during the cryo reservation process due to their reduced number of cells. PROH was used for oprotectant to freeze bovine den&embryos but turned out the L st time as the or bisected embryos. PROH was shown to be more effective to be not appropriate Yfy than glycerol or DMSO in the freezing of very early embryonic stages in the mouse, rabbit and human (22-24). A potential explanation for the abnormal pressure conditions found in Experiment 3 could have been the use of marker however, worked well in sticks for sealing the straws. This method, cryopreservation wrth lycerol as the cryoprotectant, and the batches of straws and suckers were identic 3 The abnormal pressure conditions were likely caused b interactions between PROH and liquid nitrogen. The marker sticks probably le fvt the ends at the straws leaky through which liquid nitrogen could enter and cause high ressure upon thawing. Abnormal high pressure inside the straws was not in recent experiments (unpublished foun B when the straws were heat-sealed observation). This observation shows that sealing requirements can differ in relation to the cryoprotectant used. The abnormal pressure conditions which sometimes lead to movement of the different segments inside the straws can at least partially explain the lower survival rates found in Experiment 3 compared with those found in Experiment 2. In conclusion, our study indicates that more experiments are needed to overcome the enormous sensitivity of bovine demiembryos to cryopreservation, with the goal of obtaining similarly high survival rates as those for intact embryos.
REFERENCES L&n-Jensen, H. and Willadsen S. M. Deep-freezing embryos. Theriogenology B:427-432 (1983).
of cow half and quarter
Williams,.T.J., Elsden, R. P. and Seidel Jr., G. E. Identical twin bovine pregnancres derived from bisected embryos. Theriogenology 12: 114 abstr. (1982). Williams, T. J., Elsden, R. P. and Seidel Jr., G. E. Pregnan bisected bovine embryos. Theriogenology 22:521-531(1988
OCTOBER 1991 VOL. 36 NO. 4
rates with
4.
Massip, A, Van der Zwalmen, A. and Ectors, F. Production de jumeaux monozygotes par duplication d’embryons chez les bovins. Ann. Med. Vet. 129:53-57 (1985).
5. Heyman Y. and Chesne, P. Freezing bovine embryos: survival after transfer of one half, one or two blastocysts frozen in straws.Theriogenology U:241 abstr. (1984) 6. Picard, L., King, W. A. and Betteridge, K. J. Production of sexed calves from frozen thawed embryos. Vet. Rec. m:603-608 (1985). 7. Rorie,R. W., Pendleton, R. J., Youngs, C. R. and R. A. Godke. Viability of den&embryos produced before vs. after deep freezing. Theriogenology a:192 abstr. (1986). 8. Massip, A., Van der Zwalmen, P. and Ectors F. Cryopreservation of bovine half embryos by conventional and vitrification methods. In: T. Greve (ed.), Proc. of Sym osium: Application of Egg and Embryo Technologies to Domestic Animals QRoyal Veterinary and Agricultural University, Copenhagen) (1987). 9. Niemann, H., Brem, G., Sacher, B., Smidt, D. and Krausslich, H. An approach to successful freezing of demi-embryos derived &om Day 7 bovine embryos. Theriogenology 25:519-524 (1986). 10. Niemann, H. Miiglichkeiten und Grenzen des Embryotransfers bei landwirtschaftlichen Nutztieren. Tierarztl. Umsch. &&625-633 (1986). 11. Willadsen, S. M. A method for culture of micromanipulated sheep embryos and its use to produce monozygotic twins. Nature m:298-300 (1979). 12. Niemann, H. Freezing bovine embryos: effects of a one-step addition of 1.4 M glycerol. Theriogenology 23:369-379 (1985). 13. Biery K. A. and Kraemer D. C. Comparison of Ham’s FlO, modified Whitten’s and modified Eagle’s medta supplemented with either bovine serum albumin or newborn calf serum for the in vitro culture of 8-cell mouse embryos. Theriogenology a:225 abstr. (1988). 14. Bavister, B. D. Studies on developmental blocks in cultured hamster embryos. In: B.D. Bavister (ed.), The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation In Vitro, Plenum Press, New York, 1987, pp. 219-249. 15. Wurth, Y. A. and Kruip, T. A. M. Evaluation of different media for culture of bovme demi embryos. 11th Int. Cong. Anim. Reprod. and AI (Dublm, Ireland) 1. : 484 abstr. (1988). 16. Niemann, H., Pryor, J. H. and Bondioli, K. R. Effects of slitting the zona pellucida and its subsequent sealing on freeze-thaw survival of Day 7 bovine embryos.Theriogenology a:675681 (1987). 17. Picard, L., Schneider, U., Betteridge, K. J. and King, W.A. Effects of the zona pellucida, agar-embedding and culture on the survival of micromanipulated bovine embryos after freezing and thawing. J. In Vitro Fertil. Emb. Transf.5:268-274 (1988). OCTOBER 1991 VOL. 36 NO. 4
18. Tsunoda, Y., Tokmagy, T., Okubo, Y. and Sugie, T. Beneficial effect of agar for the frozen storage of bisected embryos. Theriogenology 28: 317-322 (1987). 19. Takeda, T., Henderson, B. and Hasler J.F. Dee freezing of split and intact bovine embryos. Theriogenology a:285 abstr. Q1987). 20. Bielanski, A. and Hare, W.C.D. Survival in vitro of bovine embryos after freezing by slow cooling rates or vitrification. Theriogenology a:223 abstr. (1988). 21. Chesne, P., Heyman, Y., Chupin, D., Procureur, R. and Menezo, Y. Freezing cattle den&embryos: influence of a eriod of culture between splitting and freezing on survival. Theriogeno Pogy a:218 abstr. (1987). 22. Renard, J.P., Bui-Xuan-Nguyen and Gamier, V. Two-step freezing of twocell rabbit embryos after partial dehydration at room temperature. J. Reprod. Fertil. Z:573-580 (1984). 23. Hemandez-Ledezma, J. J. and Wright, R.W. Jr., Deep-freezing of mouse one-cell embryos and oocytes using different cryoprotectants. Theriogenology Z:735-743 (1989). 24. Siebzehnruebl, E. R., Todorow, S., Van Uem, J., Koch, R., Wildt, L. and Lang, N. Cryopreservation of human and rabbit oocytes and one-cell embryos: a corn arison of DMSO and propanediol. Hum. Reprod. &312-317 (1989 P.
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