- Email: [email protected]
freezing
Animal Reproduction Science, 13 (1987) 221-228 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
221
S u r v i v a l of Rabbit E m b r y o s after Culture or Culture/Freezing M. TECHAKUMPHU*, S. WINTENBERGER-TORRES, C. SEVELLEC and Y. MI~N]~ZO1
I.N.R.A., Station de Physiologie Animale, 78350 Jouy-en-Josas (France) II.N.S.A., Laboratoire de Biologie 406, 20 avenue A. Einstein, 69621 ViUeurbanne cddex (France) *Present address: Animal Health and Technical Service Operation, Charoen Pokphand Company, Bangkok (Thailand) (Accepted 22 August 1986)
ABSTRACT Techakumphu, M., Wintenberger-Torr~s, S., Sevellec, C. and Mdn~zo, Y., 1987. Survival of rabbit embryos after culture or culture/freezing. Anita. Reprod. Sci., 13: 221-228. Five-hundred-and-ninety-fiverabbit embryos at the 2- to 4-cell stage were cultured for 48 h to the morula stage. One-hundred-and-sixty-three embryos were transferred directly after culture while the rest (432) were frozen to - 1 9 6 ° C . The development of these embryos was tested by transfer into synchronized pseudopregnant recipients or into pseudopregnant recipients 24 h before synchrony. The results were determined at day 17 of pregnancy. The transfer of cultured embryos into synchronized recipients gave a higher survival rate than transfer into asynchronized recipients (51 vs. 15%; P < 0.05). The freezing of cultured embryos affected in vitro and in vivo development. Only 56% of the frozen-thawed morulae developed to the blastocyst stage compared with 89% in the control group (P < 0.005). The survival rate after synchronous transfer was only 14%. Our results indicate that rabbit embryos need asynchronous conditions when they are frozen and cultured. Embryo survival rate was enhanced by 38% (P<0.07) when these cultured frozenthawed embryos were transferred into pseudopregnant recipients in an earlier physiological stage ( - 2 4 h).
INTRODUCTION
In vitro culture is a method for studying the developmental capacity of the embryo before implantation. Embryos of hamsters, sheep and cattle can be cultured for a few cleavage stages while those of mice and rabbits can grow from the 1- to 2-cell to the blastocyst stage (Brinster, 1963; Krishnan and Daniel, 1967; Whitten and Biggers, 1968; Kane and Foote, 1970, 1971; Kane, 1972). These embryos can implant and develop into viable fetuses if they are transferred into suitable recipients (Chang, 1948; McLaren and Michie, 1956). However, viability after transfer depends on the time of culture (Adams, 1970; 0378-4320/87/$03.50
© 1987 Elsevier Science Publishers B.V.
222 Schneider et al., 1982; Beier et al., 1983) ; few embryos survive if that time is longer than 24 h. This may be due to retarded embryonic development, leading to asynchronization between the embryos and the maternal environment. Thus, cultured embryos are usually transferred into asynchronized recipients (Maurer et al., 1968, 1970; Binkerd and Anderson, 1979). Reports on mice and women ( Massip et al., 1984; Mohr et al., 1985 ) have shown that cultured embryos can survive and produce young after freezing to - 196 ° C, but the survival rate is generally low. This paper also reports the success of transferring rabbit embryos into synchronous and asynchronous recipients after culture/freezing. In our previous study, freezing to - 196 ° C did not modify the capacity of the embryos to develop; they survived better in synchronous than in asynchronous conditions, as also reported for non-frozen embryos (Techakumphu and Heyman, 1987; Techakumphu et al., 1987). MATERIALSAND METHODS
Embryos New Zealand or California rabbits were treated for superovulation during spring and summer with 2.0 mg of pFSH (Burns-Biotec, Omaha, U.S.A. ) and 0.33 mg ofpLH as described in our previous paper (Techakumphu et al., 1987). Twenty-four to 26 h after mating, 2- to 4-cell embryos were collected at 37 °C by flushing the oviducts with 10 ml of PBS (Whittingham, 1971) supplemented with 10% of fetal calf serum (FCS, Gibco, U.S.A.). The recovered embryos were washed once in B2 culture medium (Mdndzo, 1976). Only normal embryos with divided blastomeres were cultured directly after morphological observation.
Culture A group of 20 - 25 embryos was cultured in i ml of B2 medium supplemented with 10% of FCS in a culture dish (Nunc) at 38°C. The dish was placed in a closed dessicator and gassed with 5% CO2 + 5% 02 + 90% N2 for about 1 h. The interval between embryo collection and the beginning of culture was about 30 min. The development of the embryos was observed once after 48 h of culture.
Estimation of embryo quality after culture The embryos were selected on their morphological appearance and stage of development to a compacted morula. Non-developed and non-compacted embryos were discarded. The selected morulae were divided into two main groups. The first was used for direct transfer after 48 h of culture and the
223 second group was frozen to - 1 9 6 °C to study the additional effect of culture and freezing on embryonic development.
Freeze-thaw procedures Freeze-thaw procedures for rabbit embryos were the same as described by R e n a r d et al. (1982) for bovine embryos in straws.
Freezing. After 48 h of culture, the embryos were washed once in 1 ml of P B S + 2 0 % of F C S and later equilibrated in a cryoprotective solution of dimethylsulfoxide (DMSO: (CH~)2SO; Merck, Socolab, Paris; mol. wt: 78.13) added progressively at room temperature (20 ° C) : 0.05 M for 5 m i n , 1.0 M for 5 min, and 1.5 M for 10-15 min. This gradient was prepared from a 3 M stock solution diluted in P B S supplemented with 20% F C S ( T e c h a k u m p h u and H e y m a n , 1987). Groups of 10-12 embryos were then aspirated in small plastic straws (IMV, L'Aigle, France) with 25 zl of freezing medium (1.5 M D M S O ) and isolated with two air bubbles. Each straw was sealed with a plastic rod carrying the identification of the embryos. The straws were then placed horizontally in the cooling chamber of a programmable biological freezer (Minicool Air Liquide, Paris). The temperature of the cooling chamber was reduced from ÷ 20 to - 7 ° C by steps of 5 ° C/min. After 10 min of equilibration at - 7 ° C, seeding was induced by touching the straws with a precooled metallic rod. The straws were then slowly frozen from - 7 ° C to - 35 ° C at the rate of 0.3 ° C / m i n before plunging into liquid nitrogen. The straws were stored from I h to several months at - 196 ° C.
Thawing and diluting the cryoprotectant. Rapid thawing was achieved by transferring the straws into a water bath at 37 ° C for 1 min. The cryoprotectant was removed stepwise at room temperature with the same gradient as used for freezing b u t in reverse order. After dilution, the embryos were selected according to two criteria. The first was the morphological appearance of the embryo; embryos were considered normal if they had an intact mucin coat, zona pellucida and blastomeres. However, some with a partially damaged mucin coat were also considered as normal. The second criterion was embryo fragility; this was tested by aspirating the embryo once or twice through a micropipette with a slightly smaller diameter than that of the embryo (about 150 z m ) . Embryos with an unobservable broken zona pellucida usually do not resist aspiration. This method allowed us to limit the interval of observation and transfer when a large n u m b e r of embryos was thawed. The embryos were finally washed once in B2 medium and stored in that medium at 37°C until transfer (usually 30 min to 1 h after thawing). Seventythree frozen-thawed embryos were cultured directly to assess their development.
224
Assessment of embryo development In vitro. The frozen-thawed embryos were cultured at 37°C in I ml of B2 medium supplemented with 10% of FCS in a culture dish (Nunc) with a gas phase of 5% CO2, 5% 02 and 90% N2. The development of the frozen-thawed embryos was assessed by the formation of blastocysts after 48 h of culture. Their development was compared to that of non-frozen embryos cultured from 2- to 4-cell stages for 96 h.
In vivo. After culture or culture/freezing, only normal embryos were transferred into the oviducts of recipients ovulated at the same time as the donors or 24 h later. Ovulation was induced by mating with a vasectomized male. For transfer, we used a teflon catheter attached to a Hamilton syringe. The embryos were aspirated into the catheter with about 20 #1 of medium and air and then injected through the oviduct of the recipient at 2 cm from the infundibulum. Six embryos were usually transferred into each oviduct, making 12 embryos per recipient. The recipients were sacrificed at day 17 of gestation to determine the number of implanted and viable fetuses. The implantation and survival rates calculated were based on the number of transferred embryos.
Statistical analysis Analysis of variance after angular transformation was used to compare the implantation and survival rates per female. The percentage of in vitro development between the two different groups was compared by X2 analysis with Yates' correction. RESULTS
After 48 h of culture, 94% (856/915) of the 2- to 4-cell embryos developed into compacted morulae. One-hundred-and-sixty-three were transferred directly into synchronized or asynchronized ( - 24 h) recipients. There was no difference in pregnancy rate at day 17: 89% (8/9) for synchronous transfer and 80% (4/5) for asynchronous transfer. The implantation and survival rates of the synchronized group were higher than those of the asynchronized group (62 vs. 27%, P<0.02 and 51 vs. 15%, P<0.05, respectively) (Table 1). The survival of cultured morulae after freezing is presented in Table 2. Most of the damage occurred on the envelope: the mucin coat and zona pellucida. After 48 h of culture, the proportion of blastocysts of frozen-thawed morulae was significantly lower than that of the controls (56 vs. 89%; P < 0.005) (Table 3). The in vivo development of the frozen-thawed embryos is summarized in
225 TABLE 1 In vivo development of cultured rabbit morulae after transfer to synchronized or asynchronized recipients Synchrony recipient/donor
No. of recipients
No. of pregnancies
No. of embryos transferred
No. of implantations
No. of normal fetuses
0h -24 h
9 5
8 4
103 60
64 (62) 16 (27)**
53 (51) 9 (15)*
*P < 0.05 for comparisons with synchronous groups. **P< 0.02 for comparisons with synchronous group. ( ) percentages of survival. TABLE 2 Morphological appearance of cultured frozen-thawed rabbit morulae No. of frbzen embryos No. of recovered embryos No. of normal embryos No. of damaged embryos
432 420 (97%) 217 (52%) 203 (48%)
TABLE 3 In vitro development of cultured frozen-thawed rabbit morulae Groups
No. of embryos
No. of blastocysts after 48 h of culture
Treated embryos Controlsa
73 74
41 (56) * 66 (89)
*P < 0.005 for comparisons with controls. a2-4-cell stage embryos (24-26 h post culture) cultured for 96 h without freezing. ( ) Percentages of survival. TABLE 4 In vivo development of cultured frozen-thawed rabbit morulae after transfer to synchronized or asynchronized recipients Synchrony recipient/donor
No. of recipients
No. of pregnancies
No. of embryos transferred
No. of implantations
No. of normal fetuses
0h -24 h
6 5
3 5
65 60
22 (34) 30 (50)
9 (14) 23 (38)*
*P < 0.07 for comparisons with synchronous group. ( ) Percentages of survival.
226 Table 4. Contrary to the results on cultured embryos, the transfer of these embryos into synchronized recipients resulted in a reduced pregnancy rate. DISCUSSION With the transfer technique used, we obtained implantation and survival rates of more than 50% after the transfer of 2- to 4-cell embryos cultured for 48 h in synchronized recipients. These percentages are similar to those obtained after direct transfer in the control groups (68% of 2- to 4-cell embryos and 63% of morulae: Techakumphu et al., 1987). This shows that 48 h of culture does not modify the ability of these embryos to develop. This result is different from that in the literature which indicates considerable reduction of the in vivo development of embryos cultured for 48 h. Schneider et al. (1982) showed that the survival rate of 1-cell embryos decreased from 45 to 10% and to 5% after culture for 24, 36 and 48 h, respectively; these embryos did not survive after transfer into synchronized recipients (Maurer et al., 1968). Adams (1970) found that the survival rate decreased about 30% when the time of culture was lengthened from 24 to 48 h. A delay in the division rate and synthetic activity during culture is l~robably the cause of the low survival rate of these embryos (Maurer et al., 1970; Hahn, 1982 ). Binkerd and Anderson (1979) showed that the transfer of 2- to 4-cell embryos cultured for 70 to - 48 h in an asynchronized recipient increased the survival rate to 12% compared with 0% in synchronous transfer. The high survival rate of our embryos cultured for 48 h (50%) can be explained by favourable culture conditions which do not induce retarded development. These conditions were: - the use of B2 medium based on oviductal composition which has been proved to favour embryo development ( Mdndzo, 1976) ; fetal calf serum was added to this medium; - a culture temperature of 38°C (rabbit body temperature) instead of 37 or 37.5 ° as usually reported in the literature. We cultured for 48 h continuously and without oil; all manipulations from collection to transfer carried out at 37 ° C. Freezing after culture has a negative effect on embryo viability; the percentage of damaged embryos can reach 48%, while only 20% are found when noncultured morulae are frozen (Techakumphu and Heyman, 1987). In the present study, most of the damage occurred on the mucin coat and zona pellucida without destroying the blastomeres. Embryos with only blastomere destruction were rare. The thickness of the mucin coat might play a role by protecting the blastomeres of non-cultured morulae from osmotic shock during freezingthawing better than those of cultured morulae: the latter had a mucin coat ten times thinner than that of the non-cultured morulae. Studies on micromanipulation have shown that the zona pellucida of rabbits is mechanically less -
227 resistant t h a n t h a t of cows and sheep (Techakumphu, 1983). The zona pellucida alone does not appear to protect rabbit embryos sufficiently during freezing and thawing. The second fact t h a t might explain the damage is a structural modification of the mucin coat or the zona pellucida during culture. In rabbits we have frequently observed t h a t cultured blastocysts hatch in vitro, while in natural conditions this never occurs. The loss of zona pellucida elasticity might explain its rupture during freezing and thawing. The effects of synchronous and asynchronous transfer on the viability of cultured embryos after freezing have been studied in only two other species: mice (Massip et al., 1984) and women (Zeilmaker et al., 1984; Cohen et al., 1985; M o h r et al., 1985 ). These authors suggested t h a t the successful transfer of cultured/frozen embryos needs asynchronous conditions. Our results on the development of frozen-thawed rabbit embryos in culture support this notion. CONCLUSION The present data indicate t h a t freezing after culture m a y modify the aptitude of the embryos to develop while culture alone does not. This modification may result from retarded metabolic activity due to freezing after culture. The improvement of i m p l a n t a t i o n and survival rates after asynchronous transfer, which allows time for metabolic activity to recover, confirms this hypothesis. The cumulative effect of culture/freezing on embryo survival after transfer is still to be solved. ACKNOWLEDGEMENTS The authors wish to t h a n k A. Solari for carrying out the statistical analysis and A. Daifuku for editing the English text.
REFERENCES Adams, C.E., 1970. The developmentof rabbit eggsafter culture in vitro for 1-4 dayS.J. Embryol. Exp. Morphol., 23: 21-34. Beier, H.M., Mootz, V., Fischer, B. and StrSbele-Milller, R., 1983. Growth and differentiation of rabbit blastocysts in defined culture media. In: H.M. Beier and H.R. Lindner (Editors), Fertilization of the Human Egg in vitro. Springer-Verlag, Berlin, Heidelberg,pp. 371-386. Binkerd, P.E. and Anderson, G.B., 1979. Transfer of cultured rabbit embryos. Gamete Res., 2: 65-73. Brinster, R.L., 1963. A method for in vitro cultivation of mouse ova from two-cellto blastocyst. Exp. Cell Res., 32: 205-208. Chang, M.C., 1948. Transplantation of fertilized rabbit ova"the effect on viability of age, in vitro storage period, and storage temperature. Nature, 161: 979. Cohen, J., Simons, R.F., Edwards, R.G., Fehilly, G.B. and Fishel, S.M., 1985. Pregnancies follow-
228 ing the frozen storage of expanding human blastocysts. J. in vitro Fertil. Embryo Transf., 2: 59-64. Hahn, J., 1982. Technical aspects and perspectives of embryo transfer in laboratory animals. 2nd Congr. Int. Transfert d'Embryons chez les Mammif'eres, 20-22 Sept., Annecy, France, pp. 215-222. Kane, M.T. and Foote, R.H., 1970. Culture of two and four-cell rabbit embryos to the blastocyst stage in serum and serum extracts. Biol. Reprod., 2: 245-250. Kane, M.T. and Foote, R.H., 1971. Factors affecting blastocyst expansion of rabbit zygotes and young embryos in defined media. Biol. Reprod., 4: 41-47. Kane, M.T., 1972. Energy substrates and culture of single-cell rabbit ova to blastocysts. Nature, Lond., 238: 468-469. Krishnan, R.S. and Daniel, J.C., Jr., 1967. "Blastokinin": inducer and regulator of blastocyst development in the rabbit uterus. Science, N.Y., 158: 490-492. Massip, A., Van der Zwalmen, P., Puissant, P., Camus, F. and Leroy, F., 1984. Effects of in vitro fertilization, culture, freezing and transfer on the ability of mouse embryos to implant and survive. J. Reprod. Fertil., 71: 199-204. Maurer, R.E., Hafez, E.S.E., Ehlers, M.H. and King, J.R., 1968. Culture of two-cell rabbit eggs in chemically defined media. Exp. Cell Res., 52: 293-300. Maurer, R.R., Onuma, H. and Foote, R.H., 1970. Viability of cultured and transferred rabbit embryos. J. Reprod. Fertil., 21: 417-422. M~ndzo, Y., 1976. Milieu synth~tique pour la survie et la maturation des gametes et pour la culture de l'oeuf fdcond& C.R. Acad. Sci. Paris, S6r. D., 282: 1967-1970. McLaren, A. and Michie, D., 1956. Studies of the transfer of fertilized mouse eggs to uterine foster mothers. I. Factors affecting the implantation and survival of native transferred eggs. J. Exp. Biol., 33: 394-416. Mohr, L.R., Trounson, A. and Freemann, L., 1985. Deep-freezing and transfer of human embryos. J. in vitro Fertil. Embryo Transf., 2: 1-10. Renard, J.P., Heyman, Y. and Ozil, J.P., 1982. Congdlation de l'embryon bovin: une nouvelle mdthode de ddcongdlation pour le transfert cervical d'embryons conditionnds une seule fois en paillettes. Ann. M~d. Vdt., 126: 23-32. Schneider, U., A1-Hasani, S., Hahn, J. and Dankowski, K., 1982. Technical aspects and perspectives of embryo transfer in laboratory animals. 2e Congr. Int. Transfert d'Embryons chez les Mammif~res, 20-22 Sept., Annecy, France, p. 215 (cited by Hahn, 1982). Techakumphu, M., 1983. Recherche d'une m$thode de production de jumeaux monozygotes chez la lapine. Mdmoire de D.E.A. Physiol. Reprod., Universit~ Paris VI, Paris, 26 pp. Techakumphu, M. and Heyman, Y., 1987. Survival of frozen-thawed rabbit morulae after synchronous or asynchronous transfer. Anita. Reprod. Sci., 12: 305-312. Techakumphu, M., Wintenberger-Torr~s, S. and Sevellec, C., 1987. Survival of rabbit embryos after synchronous or asynchronous transfer. Anim. Reprod. Sci., 12: 297-304. Whitten, W.K. and Biggers, J.D., 1968. Complete development in vitro of the pre-implantation stages of the mouse in a simple chemically defined medium. J. Reprod. Fertil., 17: 399-401. Whittingham, D.G., 1971. Culture of mouse ova. J. Reprod. Fertil., Suppl., 14" 7-21. Zeilmaker, G.H., Alberda, A.T., Van Gent, I., Rijkmans, C.M.P.M. and Drogendijk, A.C., 1984. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil. Steril., 42: 293-296.
221
S u r v i v a l of Rabbit E m b r y o s after Culture or Culture/Freezing M. TECHAKUMPHU*, S. WINTENBERGER-TORRES, C. SEVELLEC and Y. MI~N]~ZO1
I.N.R.A., Station de Physiologie Animale, 78350 Jouy-en-Josas (France) II.N.S.A., Laboratoire de Biologie 406, 20 avenue A. Einstein, 69621 ViUeurbanne cddex (France) *Present address: Animal Health and Technical Service Operation, Charoen Pokphand Company, Bangkok (Thailand) (Accepted 22 August 1986)
ABSTRACT Techakumphu, M., Wintenberger-Torr~s, S., Sevellec, C. and Mdn~zo, Y., 1987. Survival of rabbit embryos after culture or culture/freezing. Anita. Reprod. Sci., 13: 221-228. Five-hundred-and-ninety-fiverabbit embryos at the 2- to 4-cell stage were cultured for 48 h to the morula stage. One-hundred-and-sixty-three embryos were transferred directly after culture while the rest (432) were frozen to - 1 9 6 ° C . The development of these embryos was tested by transfer into synchronized pseudopregnant recipients or into pseudopregnant recipients 24 h before synchrony. The results were determined at day 17 of pregnancy. The transfer of cultured embryos into synchronized recipients gave a higher survival rate than transfer into asynchronized recipients (51 vs. 15%; P < 0.05). The freezing of cultured embryos affected in vitro and in vivo development. Only 56% of the frozen-thawed morulae developed to the blastocyst stage compared with 89% in the control group (P < 0.005). The survival rate after synchronous transfer was only 14%. Our results indicate that rabbit embryos need asynchronous conditions when they are frozen and cultured. Embryo survival rate was enhanced by 38% (P<0.07) when these cultured frozenthawed embryos were transferred into pseudopregnant recipients in an earlier physiological stage ( - 2 4 h).
INTRODUCTION
In vitro culture is a method for studying the developmental capacity of the embryo before implantation. Embryos of hamsters, sheep and cattle can be cultured for a few cleavage stages while those of mice and rabbits can grow from the 1- to 2-cell to the blastocyst stage (Brinster, 1963; Krishnan and Daniel, 1967; Whitten and Biggers, 1968; Kane and Foote, 1970, 1971; Kane, 1972). These embryos can implant and develop into viable fetuses if they are transferred into suitable recipients (Chang, 1948; McLaren and Michie, 1956). However, viability after transfer depends on the time of culture (Adams, 1970; 0378-4320/87/$03.50
© 1987 Elsevier Science Publishers B.V.
222 Schneider et al., 1982; Beier et al., 1983) ; few embryos survive if that time is longer than 24 h. This may be due to retarded embryonic development, leading to asynchronization between the embryos and the maternal environment. Thus, cultured embryos are usually transferred into asynchronized recipients (Maurer et al., 1968, 1970; Binkerd and Anderson, 1979). Reports on mice and women ( Massip et al., 1984; Mohr et al., 1985 ) have shown that cultured embryos can survive and produce young after freezing to - 196 ° C, but the survival rate is generally low. This paper also reports the success of transferring rabbit embryos into synchronous and asynchronous recipients after culture/freezing. In our previous study, freezing to - 196 ° C did not modify the capacity of the embryos to develop; they survived better in synchronous than in asynchronous conditions, as also reported for non-frozen embryos (Techakumphu and Heyman, 1987; Techakumphu et al., 1987). MATERIALSAND METHODS
Embryos New Zealand or California rabbits were treated for superovulation during spring and summer with 2.0 mg of pFSH (Burns-Biotec, Omaha, U.S.A. ) and 0.33 mg ofpLH as described in our previous paper (Techakumphu et al., 1987). Twenty-four to 26 h after mating, 2- to 4-cell embryos were collected at 37 °C by flushing the oviducts with 10 ml of PBS (Whittingham, 1971) supplemented with 10% of fetal calf serum (FCS, Gibco, U.S.A.). The recovered embryos were washed once in B2 culture medium (Mdndzo, 1976). Only normal embryos with divided blastomeres were cultured directly after morphological observation.
Culture A group of 20 - 25 embryos was cultured in i ml of B2 medium supplemented with 10% of FCS in a culture dish (Nunc) at 38°C. The dish was placed in a closed dessicator and gassed with 5% CO2 + 5% 02 + 90% N2 for about 1 h. The interval between embryo collection and the beginning of culture was about 30 min. The development of the embryos was observed once after 48 h of culture.
Estimation of embryo quality after culture The embryos were selected on their morphological appearance and stage of development to a compacted morula. Non-developed and non-compacted embryos were discarded. The selected morulae were divided into two main groups. The first was used for direct transfer after 48 h of culture and the
223 second group was frozen to - 1 9 6 °C to study the additional effect of culture and freezing on embryonic development.
Freeze-thaw procedures Freeze-thaw procedures for rabbit embryos were the same as described by R e n a r d et al. (1982) for bovine embryos in straws.
Freezing. After 48 h of culture, the embryos were washed once in 1 ml of P B S + 2 0 % of F C S and later equilibrated in a cryoprotective solution of dimethylsulfoxide (DMSO: (CH~)2SO; Merck, Socolab, Paris; mol. wt: 78.13) added progressively at room temperature (20 ° C) : 0.05 M for 5 m i n , 1.0 M for 5 min, and 1.5 M for 10-15 min. This gradient was prepared from a 3 M stock solution diluted in P B S supplemented with 20% F C S ( T e c h a k u m p h u and H e y m a n , 1987). Groups of 10-12 embryos were then aspirated in small plastic straws (IMV, L'Aigle, France) with 25 zl of freezing medium (1.5 M D M S O ) and isolated with two air bubbles. Each straw was sealed with a plastic rod carrying the identification of the embryos. The straws were then placed horizontally in the cooling chamber of a programmable biological freezer (Minicool Air Liquide, Paris). The temperature of the cooling chamber was reduced from ÷ 20 to - 7 ° C by steps of 5 ° C/min. After 10 min of equilibration at - 7 ° C, seeding was induced by touching the straws with a precooled metallic rod. The straws were then slowly frozen from - 7 ° C to - 35 ° C at the rate of 0.3 ° C / m i n before plunging into liquid nitrogen. The straws were stored from I h to several months at - 196 ° C.
Thawing and diluting the cryoprotectant. Rapid thawing was achieved by transferring the straws into a water bath at 37 ° C for 1 min. The cryoprotectant was removed stepwise at room temperature with the same gradient as used for freezing b u t in reverse order. After dilution, the embryos were selected according to two criteria. The first was the morphological appearance of the embryo; embryos were considered normal if they had an intact mucin coat, zona pellucida and blastomeres. However, some with a partially damaged mucin coat were also considered as normal. The second criterion was embryo fragility; this was tested by aspirating the embryo once or twice through a micropipette with a slightly smaller diameter than that of the embryo (about 150 z m ) . Embryos with an unobservable broken zona pellucida usually do not resist aspiration. This method allowed us to limit the interval of observation and transfer when a large n u m b e r of embryos was thawed. The embryos were finally washed once in B2 medium and stored in that medium at 37°C until transfer (usually 30 min to 1 h after thawing). Seventythree frozen-thawed embryos were cultured directly to assess their development.
224
Assessment of embryo development In vitro. The frozen-thawed embryos were cultured at 37°C in I ml of B2 medium supplemented with 10% of FCS in a culture dish (Nunc) with a gas phase of 5% CO2, 5% 02 and 90% N2. The development of the frozen-thawed embryos was assessed by the formation of blastocysts after 48 h of culture. Their development was compared to that of non-frozen embryos cultured from 2- to 4-cell stages for 96 h.
In vivo. After culture or culture/freezing, only normal embryos were transferred into the oviducts of recipients ovulated at the same time as the donors or 24 h later. Ovulation was induced by mating with a vasectomized male. For transfer, we used a teflon catheter attached to a Hamilton syringe. The embryos were aspirated into the catheter with about 20 #1 of medium and air and then injected through the oviduct of the recipient at 2 cm from the infundibulum. Six embryos were usually transferred into each oviduct, making 12 embryos per recipient. The recipients were sacrificed at day 17 of gestation to determine the number of implanted and viable fetuses. The implantation and survival rates calculated were based on the number of transferred embryos.
Statistical analysis Analysis of variance after angular transformation was used to compare the implantation and survival rates per female. The percentage of in vitro development between the two different groups was compared by X2 analysis with Yates' correction. RESULTS
After 48 h of culture, 94% (856/915) of the 2- to 4-cell embryos developed into compacted morulae. One-hundred-and-sixty-three were transferred directly into synchronized or asynchronized ( - 24 h) recipients. There was no difference in pregnancy rate at day 17: 89% (8/9) for synchronous transfer and 80% (4/5) for asynchronous transfer. The implantation and survival rates of the synchronized group were higher than those of the asynchronized group (62 vs. 27%, P<0.02 and 51 vs. 15%, P<0.05, respectively) (Table 1). The survival of cultured morulae after freezing is presented in Table 2. Most of the damage occurred on the envelope: the mucin coat and zona pellucida. After 48 h of culture, the proportion of blastocysts of frozen-thawed morulae was significantly lower than that of the controls (56 vs. 89%; P < 0.005) (Table 3). The in vivo development of the frozen-thawed embryos is summarized in
225 TABLE 1 In vivo development of cultured rabbit morulae after transfer to synchronized or asynchronized recipients Synchrony recipient/donor
No. of recipients
No. of pregnancies
No. of embryos transferred
No. of implantations
No. of normal fetuses
0h -24 h
9 5
8 4
103 60
64 (62) 16 (27)**
53 (51) 9 (15)*
*P < 0.05 for comparisons with synchronous groups. **P< 0.02 for comparisons with synchronous group. ( ) percentages of survival. TABLE 2 Morphological appearance of cultured frozen-thawed rabbit morulae No. of frbzen embryos No. of recovered embryos No. of normal embryos No. of damaged embryos
432 420 (97%) 217 (52%) 203 (48%)
TABLE 3 In vitro development of cultured frozen-thawed rabbit morulae Groups
No. of embryos
No. of blastocysts after 48 h of culture
Treated embryos Controlsa
73 74
41 (56) * 66 (89)
*P < 0.005 for comparisons with controls. a2-4-cell stage embryos (24-26 h post culture) cultured for 96 h without freezing. ( ) Percentages of survival. TABLE 4 In vivo development of cultured frozen-thawed rabbit morulae after transfer to synchronized or asynchronized recipients Synchrony recipient/donor
No. of recipients
No. of pregnancies
No. of embryos transferred
No. of implantations
No. of normal fetuses
0h -24 h
6 5
3 5
65 60
22 (34) 30 (50)
9 (14) 23 (38)*
*P < 0.07 for comparisons with synchronous group. ( ) Percentages of survival.
226 Table 4. Contrary to the results on cultured embryos, the transfer of these embryos into synchronized recipients resulted in a reduced pregnancy rate. DISCUSSION With the transfer technique used, we obtained implantation and survival rates of more than 50% after the transfer of 2- to 4-cell embryos cultured for 48 h in synchronized recipients. These percentages are similar to those obtained after direct transfer in the control groups (68% of 2- to 4-cell embryos and 63% of morulae: Techakumphu et al., 1987). This shows that 48 h of culture does not modify the ability of these embryos to develop. This result is different from that in the literature which indicates considerable reduction of the in vivo development of embryos cultured for 48 h. Schneider et al. (1982) showed that the survival rate of 1-cell embryos decreased from 45 to 10% and to 5% after culture for 24, 36 and 48 h, respectively; these embryos did not survive after transfer into synchronized recipients (Maurer et al., 1968). Adams (1970) found that the survival rate decreased about 30% when the time of culture was lengthened from 24 to 48 h. A delay in the division rate and synthetic activity during culture is l~robably the cause of the low survival rate of these embryos (Maurer et al., 1970; Hahn, 1982 ). Binkerd and Anderson (1979) showed that the transfer of 2- to 4-cell embryos cultured for 70 to - 48 h in an asynchronized recipient increased the survival rate to 12% compared with 0% in synchronous transfer. The high survival rate of our embryos cultured for 48 h (50%) can be explained by favourable culture conditions which do not induce retarded development. These conditions were: - the use of B2 medium based on oviductal composition which has been proved to favour embryo development ( Mdndzo, 1976) ; fetal calf serum was added to this medium; - a culture temperature of 38°C (rabbit body temperature) instead of 37 or 37.5 ° as usually reported in the literature. We cultured for 48 h continuously and without oil; all manipulations from collection to transfer carried out at 37 ° C. Freezing after culture has a negative effect on embryo viability; the percentage of damaged embryos can reach 48%, while only 20% are found when noncultured morulae are frozen (Techakumphu and Heyman, 1987). In the present study, most of the damage occurred on the mucin coat and zona pellucida without destroying the blastomeres. Embryos with only blastomere destruction were rare. The thickness of the mucin coat might play a role by protecting the blastomeres of non-cultured morulae from osmotic shock during freezingthawing better than those of cultured morulae: the latter had a mucin coat ten times thinner than that of the non-cultured morulae. Studies on micromanipulation have shown that the zona pellucida of rabbits is mechanically less -
227 resistant t h a n t h a t of cows and sheep (Techakumphu, 1983). The zona pellucida alone does not appear to protect rabbit embryos sufficiently during freezing and thawing. The second fact t h a t might explain the damage is a structural modification of the mucin coat or the zona pellucida during culture. In rabbits we have frequently observed t h a t cultured blastocysts hatch in vitro, while in natural conditions this never occurs. The loss of zona pellucida elasticity might explain its rupture during freezing and thawing. The effects of synchronous and asynchronous transfer on the viability of cultured embryos after freezing have been studied in only two other species: mice (Massip et al., 1984) and women (Zeilmaker et al., 1984; Cohen et al., 1985; M o h r et al., 1985 ). These authors suggested t h a t the successful transfer of cultured/frozen embryos needs asynchronous conditions. Our results on the development of frozen-thawed rabbit embryos in culture support this notion. CONCLUSION The present data indicate t h a t freezing after culture m a y modify the aptitude of the embryos to develop while culture alone does not. This modification may result from retarded metabolic activity due to freezing after culture. The improvement of i m p l a n t a t i o n and survival rates after asynchronous transfer, which allows time for metabolic activity to recover, confirms this hypothesis. The cumulative effect of culture/freezing on embryo survival after transfer is still to be solved. ACKNOWLEDGEMENTS The authors wish to t h a n k A. Solari for carrying out the statistical analysis and A. Daifuku for editing the English text.
REFERENCES Adams, C.E., 1970. The developmentof rabbit eggsafter culture in vitro for 1-4 dayS.J. Embryol. Exp. Morphol., 23: 21-34. Beier, H.M., Mootz, V., Fischer, B. and StrSbele-Milller, R., 1983. Growth and differentiation of rabbit blastocysts in defined culture media. In: H.M. Beier and H.R. Lindner (Editors), Fertilization of the Human Egg in vitro. Springer-Verlag, Berlin, Heidelberg,pp. 371-386. Binkerd, P.E. and Anderson, G.B., 1979. Transfer of cultured rabbit embryos. Gamete Res., 2: 65-73. Brinster, R.L., 1963. A method for in vitro cultivation of mouse ova from two-cellto blastocyst. Exp. Cell Res., 32: 205-208. Chang, M.C., 1948. Transplantation of fertilized rabbit ova"the effect on viability of age, in vitro storage period, and storage temperature. Nature, 161: 979. Cohen, J., Simons, R.F., Edwards, R.G., Fehilly, G.B. and Fishel, S.M., 1985. Pregnancies follow-
228 ing the frozen storage of expanding human blastocysts. J. in vitro Fertil. Embryo Transf., 2: 59-64. Hahn, J., 1982. Technical aspects and perspectives of embryo transfer in laboratory animals. 2nd Congr. Int. Transfert d'Embryons chez les Mammif'eres, 20-22 Sept., Annecy, France, pp. 215-222. Kane, M.T. and Foote, R.H., 1970. Culture of two and four-cell rabbit embryos to the blastocyst stage in serum and serum extracts. Biol. Reprod., 2: 245-250. Kane, M.T. and Foote, R.H., 1971. Factors affecting blastocyst expansion of rabbit zygotes and young embryos in defined media. Biol. Reprod., 4: 41-47. Kane, M.T., 1972. Energy substrates and culture of single-cell rabbit ova to blastocysts. Nature, Lond., 238: 468-469. Krishnan, R.S. and Daniel, J.C., Jr., 1967. "Blastokinin": inducer and regulator of blastocyst development in the rabbit uterus. Science, N.Y., 158: 490-492. Massip, A., Van der Zwalmen, P., Puissant, P., Camus, F. and Leroy, F., 1984. Effects of in vitro fertilization, culture, freezing and transfer on the ability of mouse embryos to implant and survive. J. Reprod. Fertil., 71: 199-204. Maurer, R.E., Hafez, E.S.E., Ehlers, M.H. and King, J.R., 1968. Culture of two-cell rabbit eggs in chemically defined media. Exp. Cell Res., 52: 293-300. Maurer, R.R., Onuma, H. and Foote, R.H., 1970. Viability of cultured and transferred rabbit embryos. J. Reprod. Fertil., 21: 417-422. M~ndzo, Y., 1976. Milieu synth~tique pour la survie et la maturation des gametes et pour la culture de l'oeuf fdcond& C.R. Acad. Sci. Paris, S6r. D., 282: 1967-1970. McLaren, A. and Michie, D., 1956. Studies of the transfer of fertilized mouse eggs to uterine foster mothers. I. Factors affecting the implantation and survival of native transferred eggs. J. Exp. Biol., 33: 394-416. Mohr, L.R., Trounson, A. and Freemann, L., 1985. Deep-freezing and transfer of human embryos. J. in vitro Fertil. Embryo Transf., 2: 1-10. Renard, J.P., Heyman, Y. and Ozil, J.P., 1982. Congdlation de l'embryon bovin: une nouvelle mdthode de ddcongdlation pour le transfert cervical d'embryons conditionnds une seule fois en paillettes. Ann. M~d. Vdt., 126: 23-32. Schneider, U., A1-Hasani, S., Hahn, J. and Dankowski, K., 1982. Technical aspects and perspectives of embryo transfer in laboratory animals. 2e Congr. Int. Transfert d'Embryons chez les Mammif~res, 20-22 Sept., Annecy, France, p. 215 (cited by Hahn, 1982). Techakumphu, M., 1983. Recherche d'une m$thode de production de jumeaux monozygotes chez la lapine. Mdmoire de D.E.A. Physiol. Reprod., Universit~ Paris VI, Paris, 26 pp. Techakumphu, M. and Heyman, Y., 1987. Survival of frozen-thawed rabbit morulae after synchronous or asynchronous transfer. Anita. Reprod. Sci., 12: 305-312. Techakumphu, M., Wintenberger-Torr~s, S. and Sevellec, C., 1987. Survival of rabbit embryos after synchronous or asynchronous transfer. Anim. Reprod. Sci., 12: 297-304. Whitten, W.K. and Biggers, J.D., 1968. Complete development in vitro of the pre-implantation stages of the mouse in a simple chemically defined medium. J. Reprod. Fertil., 17: 399-401. Whittingham, D.G., 1971. Culture of mouse ova. J. Reprod. Fertil., Suppl., 14" 7-21. Zeilmaker, G.H., Alberda, A.T., Van Gent, I., Rijkmans, C.M.P.M. and Drogendijk, A.C., 1984. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil. Steril., 42: 293-296.