29, 485492
CRYOBIOLOGY
(1992)
In Vitro Fertilization
E. FUKU,”
T. KOJIMA,t
and Development Bovine Oocytes
of Frozen-Thawed
Y. SHIOYA,‘F G. J. MARCUS,+
AND
B. R. DOWNEY”,’
*Department of Animal Science, Macdonald Campus, McGill University, Ste. Anne de Be//ewe, Quebec, Canada H9X lC0; fDepartment of Animal Reproduction. National Institute of Animal Industry, Tsukuba-Norindanchi, P.O. Box 5, Ibaraki, 305, Japan; and SCentre for Food and Animal Research Agriculture Canada, Ottawa, Ontario, Canada KIA OC6 Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%). but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a cl 1992 Academic Preab. Inc. recipient heifer resulted in pregnancy and the birth of twin calves.
Success in the cryopreservation of em- new interest in the preservation of oocytes bryos was first achieved in the pioneering at low temperatures. Thus far, some success has been work of Whittingham ef al. (29), with the mouse, in the United States, and indepen- achieved with human oocytes and with dently by Wilmut (30) in Great Britain. The mouse oocytes. Trounson (26) found that protocols developed by these workers have vitrification resulted in the greatest oocyte served as the basis for cryopreservation of survival and subsequent development in the embryos of most species and have led to vitro in the human. In early studies several widespread exploitation of cryopreservainvestigators obtained normal young from tion as a routine practice in the embryo- IVF of ovulated mouse oocytes frozen transfer industry. Hitherto, procedures for slowly using DMSO, albeit with low cryothe cryopreservation of oocytes have not survival and fertilization rates (16, 25, 26). been developed to the same extent, partic- Recently, Kono et al. (12) and Nakagata ularly in the case of farm animals, due to (15, 16) reported that mouse oocytes also factors such as the generally lower yield of can be cryopreserved using vitrification viable embryos resulting from in vitro mat- and retain competence to develop into noruration (IVM), fertilization (IVF), and sub- mal offspring following IVF-IVC and emsequent culture (IVC) as well as a lack of bryo transfer. Ko and Threlfall (10) reeconomic incentives. However, the recent ported that mouse oocytes can be slowly rapid improvement of IVM-IVF-IVC tech- frozen and preserved at - 196°C using 1,2niques in the bovine (6, 25) has generated propanediol as a cryoprotectant. Hitherto, however, limited progress in the cryopreservation of oocytes of farm animals has Received December 10, 1991; accepted February been achieved (14, 31) and production of 24, 1992. offspring has not been reported. In the cur’ To whom reprint requests should he addressed. 485 001 I-2240/92 $5.00 Copyright 0 1992 by Academic Press. Lnc All rights of reproduction in any form reserved.
486
FUKU
ET AL.
oocytes into 0.25 ml French straws [I.M.V., L’Aigle, France]). The ends of the straws were heat-sealed and the straws placed horizontally in the ethanol bath of a programmable freezer (MPF-40, Tokyo MATERIALS AND METHODS Rikakikai, Japan) kept at - 8°C. After 2 min, ice formation was induced by touching Oocytes the straw with forceps cooled to - 196°C in The cumulus-oocyte complex classifica- liquid nitrogen. When seeding of all straws tion and IVM-IVF-IVC procedures used in was complete, the bath was held at -8°C these experiments were essentially the for a further 10 min and then cooled at same as described previously by Shioya et 0.3”Cmin. When the bath temperature al. (24). After collection from abattoir ova- reached -4O”C, the straws were plunged ries, bovine cumulus-oocyte complexes into liquid nitrogen. After storage for l-2 were classified according to their morpho- weeks, straws were rewarmed by holding in logical characteristics, as follows: air for 5 s and then immersing them in water Class A had a compact, dense cumulus at 22°C. The straws were cut and the concell layer; Class B had a compact but sparse tents placed for 5 min in a watch glass in cumulus cell layer (B,) or was partially de- M199a containing 1.O M propanediol and nuded (B,)-the latter were used for oocyte 0.5 M sucrose and then transferred into 0.5 evaluation, as described below; Class C M sucrose in M199a for another 5 min to oocytes were devoid of cumulus cells and dilute the cryoprotectant. were discarded. The medium used for vitrification was In the first experiment, bovine cumulus- DAP 213 (16), a mixture of 2.0 M dimethyl oocyte complexes, at the germinal vesicle sulfoxide (DMSO, Dojin Chemical Inc., (GV) stage, were cryopreserved directly. In Kumamoto, Japan), 1.0 M acetamide the second experiment, cumulus-oocyte (Sigma), and 3.0 M propanediol in M199a. complexes were cultured for 21-26 h in 100 Cumulus-oocyte complexes were imp.1droplets of maturation medium (25 mM mersed in DAP213 at room temperature, Hepes-buffered TCM- 199 with 10% calf se- loaded into 0.25-ml straws, and plunged dirum and antibiotics, see Ref. (24)) under rectly into liquid nitrogen, the whole procemineral oil (Squibb and Sons, Inc., Prince- dure taking less than 1 min. After storage ton, NJ) at 39°C in 5% CO, in air and then for l-2 weeks, the straws were warmed by frozen. holding in air for 5 s and then immersing them in water at 22°C. The contents were Freezing and Thawing immediately diluted with a single addition Two different procedures, slow freezing of 0.5 M sucrose in M199a for 5 min and and vitrification, applied to GV-stage and then expelled into M199a containing 0.5 M to in vitro matured oocytes were compared. sucrose in a watch glass and held for 5 min. In the slow freezing, the cumulus-oocyte In Vitro Fertilization complexes were immersed directly into 2.0 The basic medium (Medium BO) used for M 1,2-propanediol (Sigma Chemical Co., St. Louis, MO) in Hepes-buffered TCM- the treatment of spermatozoa and the fer199 (GIBCO, Grand Island, NY) supple- tilization of oocytes was that used by mented with 0.4% bovine serum albumin Brackett and Oliphant (2) for the fertiliza(BSA, crystallized, Sigma) (- M199a), at tion of rabbit eggs in vitro. Two or three ca. 25°C and equilibrated for 10 to 20 min 0.5-ml straws of frozen semen obtained (including the time required to load the from a single Japanese black bull were rent study, two different freezing methods (vitrification and slow freezing), applied to immature and in vitro matured bovine oocytes, were compared.
DEVELOPMENT
OF FROZEN-THAWED
thawed in water at 30°C. Spermatozoa were washed 2x by suspension and centrifugation at 500g for 5 min in Medium BO supplemented with 10 mM caffeine-sodium benzoate (Sigma) without BSA (Caff-BO). The pellet was resuspended in 0.5 to 1.Oml Caff-BO and the sperm concentration measured with a haemocytometer and adjusted to 107/ml. The suspension was then diluted 1:1 with Medium BO supplemented with 20 mg/ml crystallized BSA (BSA-BO) and 5 ~1 heparin/ml (Novo-Heparin 1000, 1000 U/ml Novo Industry A/S, Japan). Droplets (0.1 ml) of this sperm suspension (5 x lo6 sperm/ml) were incubated for 15 min at 39°C in 5% CO, in humid air. Thawed GV-stage cumulus-oocyte complexes, cultured for 21-26 h or complexes frozen after maturation culture, were transferred into sperm droplets for fertilization. In addition, unfrozen cumulus-oocyte complexes, matured in vitro, were placed in sperm droplets, as controls. Sperm and cumulus-oocyte complexes were incubated for 6 h at 39°C in 5% CO* in air. After washing 4~ in culture medium, the cumulus-oocyte complexes were transferred onto a proliferating monolayer of cumulus cells in 100 ~1 droplets of culture medium (25 mM Hepes-buffered TCM-199 supplemented with 10% calf serum and antibiotics, see Ref. (24)) under mineral oil at 39°C in 5% CO2 in air, for assessment of cleavage and development.
487
OOCYTES
stained with 1% orcein in 45% (v/v) aqueous acetic acid and examined at 1000X with a phase-contrast microscope (1, 17). Eggs containing both female and male pronuclei were considered to be fertilized and were deemed normal or polyspermic according to the number of pronuclei in the cytoplasm. Development to the two-cell stage was assessed 40 h after placing oocytes from class A or B cumulus complexes with sperm. The developing embryos were cultured for an additional 5 days to evaluate their capacity to develop into morulae or blastocysts. Four embryos (two morulae and two blastocysts) derived from oocytes slowly frozen after IVM were placed in Hepesbuffered TCM199 supplemented with 10% calf serum, loaded into an 0.25-ml straw, and deposited nonsurgically in the left uterine horn, ipsilateral to a corpus luteum, in a Holstein recipient at Day 7 of the cycle (Day 0 = onset of estrus). The progress of pregnancy was monitored periodically by ultrasonic scanning. Statistical
Analysis
Data from eight replicate experiments were pooled and the treatment effects were compared by x2 analysis. RESULTS
Survival
of Cryopreserved
Oocytes
In preliminary experiments, oocytes frozen in the presence of 2M propanediol at Cryosurvival of oocytes was assessed7 h O.l”C/min to -40°C or at O.S”C/min to after thawing, using a stereomicroscope to -80°C survived poorly and none develcount the numbers of normal and pycnotic- oped to the two-cell stage after IVF (data appearing oocytes (the latter were assumed not shown). Recovery and morphological to be degenerate). To assess fertilization, survival of oocytes cryopreserved by slow class B, oocyte complexes were retrieved freezing (0.3”/min) and by vitrification are from the culture dishes 19-20 h after insem- compared in Table 1. Nearly 90% (1537/ ination and cumulus cells and spermatozoa 1764) of all cryopreserved oocytes were reattached to the zona pellucida removed covered. The survival rates (calculated with 0.1% hyaluronidase (Sigma). The from results pooled for A and B classes of oocytes were then fixed in 1:3 acetic acid: cumulus investment) were greater (P < ethanol for 24-28 h at room temnerature. , 0.05) after slow freezing than after vitrificaA Oocyte Evaluation
488
FUKU ET AL. TABLE 1 Effect of Freezing of Bovine Oocytes on Their Recovery and Morphological Survival* after Freezing-Thawing
Group GV, vitrified GV, frozen slowly IVM, vitrified IVM, frozen slowly
Class of cumulus investment?
No. of oocytes frozen+
No. (%) of oocytes recovered
No. (%) with normal morphology*
A B A B A B A B
120 176 129 135 351 258 325 270
100 (83.3) 160 (90.9) 107 (82.9) 121 (89.6) 316 (90.0) 226 (87.6) 269 (82.8) 238 (88.1)
13 (13.0)” 25 (15.6)” 31 (29.0)b 39 (32.2)b 45 (14.2)” 31 (13.7)O 148 (55.0)’ 122 (51.3)
* Assessed 7 h after thawing. t Class A had a compact, dense cumulus cell layer; Class B had a compact but sparse cumulus cell layer. $ GV and IVM data pooled from 3 and 5 replicates, respectively. a,b,cWithin columns, values with different superscripts are different (P < 0.05).
tion both in GV-stage oocytes (30.7 vs 14.6%) and in oocytes matured in vitro (53.3 vs 14.0%). Fertilization of Cryopreserved Oocytes Fertilization data are shown in Table 2. Oocytes frozen slowly after IVM were less capable of being fertilized (5 1%) than unfrozen, control oocytes (76%), but were more capable than vitrified oocytes or oocytes frozen prior to maturation (P < 0.05).
Development Potential of Cryopreserved Oocytes Table 3 shows the proportions of oocytes that cleaved and that developed to the morula/blastocyst stage. The proportion of cryopreserved oocytes which developed into two-cell embryos was greatest among those that had been frozen slowly after IVM (16%)) but lower than among unfrozen controls (40%, P < 0.05). However, there was no difference between unfrozen oocytes and slowly frozen IVM oocytes in
TABLE 2 The Fertilization of Cryopreserved Bovine Oocytes No. of oocytes fertilizedt
Group
No. of oocytes*
Total (%)
Normal
Polyspermic
GV, vitrified GV, frozen slowly IVM, vitrified IVM, frozen slowly IVM, control, not frozen
26 22 43 51 91
2 (7.7)” 2 (9.1)” 5 (11.6)” 26 (51.O)b 69 (75.8)
0 1 2 12 48
2 I 3 14 21
* GV and IVM data pooled from 3 and 5 replicates, respectively. The values are the total numbers of oocytes with normal post-thaw morphology. t Oocytes with male and female pronuclei. Normal and polyspermic fertilization were indicated by the presence of 2 and >2 pronuclei, respectively. o.bx Within columns, values with different superscripts are different (P < 0.05).
DEVELOPMENT
OF FROZEN-THAWED
489
OOCYTES
TABLE 3 Development Capacity of Cryopreserved Bovine Oocytes in Vitro Class of cumulus investment*
Group
No. of oocytes fertilized?
No. (%) two-cell embryos
No. of morulae (M) and blastocyst (B) {% of two-cell embryos}
I (1.1)” 1 (0.7)” l(1.1)” 2 (1.9)” 14 (4.9) 9 (4.2) 35 (16.2)b 26 (15.7)b 35 (41.7)’ 52 (38.0)’
O{ 0%) O{ O%} O{ 0%) GV, frozen slowly O{ O%} IVM, vitrified O{ 0%) B O{ 0%) A IVM, frozen slowly 4 (2B, 2M) {11.4%}$ B 2 (IB, IM) {7.7%} A IVM, control, not frozen 5 (5B) { 14.3%) B 5 (5B) {9.6%} * Class A had a compact, dense cumulus cell layer; Class B had a compact but sparse cumulus cell layer. t GV and IVM data were pooled from 3 and 5 replicates, respectively. $ Nonsurgical transfer of 2 B and 2 M into a recipient heifer resulted in a confirmed pregnancy. rr~h~c Within columns, values with different superscripts are different (P < 0.05). A B A B A
GV, vitrified
the proportions that developed into morulae or blastocysts after IVF (11.5 vs 9.8%). The class of cumulus investment of the oocytes at the time of their harvest from the ovary had no discernible effect on the developmental potential of the cryopreserved oocytes. Two morulae and two blastocysts that had developed from IVM oocytes, slowly frozen, thawed and fertilized in vitro, all transferred into a single Holstein recipient, produced a pregnancy. On April 3, 1992, twin calves were born; the female and male weighed 32.7 and 51.2 kg, respectively Fig. 1). DISCUSSION
Recently, Hernandez-Ledezma and Wright (7) reported that the use of propanediol instead of glycerol or DMSO significantly improved survival and development of cryopreserved mouse oocytes to the two-cell stage. Nakagata (15), using the medium of Rall and Fahy (21), found that ca. 88% of vitrified mouse oocytes were morphologically normal, 80% of these developed to the two-cell stage and, following
94 148 90 106 286 215 216 166 84 137
transfer, resulted in a pregnancy rate of 46%. However, inasmuch as oocytes and embryos at various stages of development differ both physiologically and morphologically (8, 9, 13), freezing techniques developed for the one may not be suitable for the other. Thus, Heyman et al. (8) reported that very few bovine oocytes (6%) matured in vitro after rapid freezing and thawing. In the present study also, ca. 30% of oocytes frozen slowly at the GV stage survived (Table l), but fewer than 5% underwent germinal vesicle breakdown (GVBD) and polar body formation (data not shown). Moreover, only three (1.5%) of these cleaved after IVF and none developed into morulae or blastocysts (Table 3). These findings contrast with those of Schroeder et al. (23) who found that GV-stage mouse oocytes survived slow freezing to -80°C 95% underwent maturation in vitro as indicated by GVBD and polar body formation (90%), but, as in the present study, few cleaved and none developed into blastocysts. This suggests that immature bovine ova are more sensitive to cryoprocessing than GVstage mouse oocytes. Sathananthan et al. (22) found that, in
490
FUKU ET AL.
FIG. 1. Twin calves (Holstein x Japanese Black) born from frozen-thawed oocytes, matured, fertilized, and cultured in vitro to morulaeMastocysts.
mouse oocytes, spindle microtubule reorganization after GVBD is particularly sensitive to cold and is readily damaged by exposure to low temperatures, the damage becoming apparent only at the time of the first mitotic division. This explains the finding that few bovine oocytes matured after cryopreservation at the GV stage and even fewer cleaved after IVF. However, Downs et al. (5) and Schroeder et al. (23) found that when meiosis was arrested with isobutyl-3-methylxanthine before cryopreservation, development was significantly improved after freezing and thawing. Exploration of such an approach should form the basis of future studies and may result in sufficiently high survival to permit the routine use of GV-stage oocytes. The finding that the cleavage rate after IVF of mature oocytes frozen slowly was higher than that of other treatment groups agrees with the similar findings of Schroeder et al. (23). Moreover, the potential of two-cell embryos derived from frozen oocytes to develop into morulae or blastocysts was not different from that of
embryos from unfrozen control oocytes. In both the present study and that of Schroeder et af. (23), the dramatic change in freezability was associated with meiotic maturation in vivo or in vitro. Moreover, the damaging effects of freezing-thawing were apparent only up to the two-cell stage but, past this hurdle, did not affect further development to morulae or blastocysts. Although mouse and human oocytes have been cryopreserved successfully by vitrification (15, 26), in the present study, vitrification applied to either immature or in vitro matured bovine oocytes resulted in very poor survival and cleavage and no development into morulae or blastocysts. Consequently, slow freezing to -40°C at 0.3”Clmin appears to be the preferred procedure for cryopreservation of bovine oocytes. Nevertheless, the nature of the effects of vitrification on bovine oocytes should be investigated as this relatively new approach is simple and easily applied under field conditions. In contrast to the observations of Pellicer er al. (19) and Schroeder ec al. (23) that
DEVELOPMENT
OF
FROZEN-THAWED
greater survival was associated with more layers of cumulus cells in frozen-thawed rat and mouse oocytes, we found no differences in survival, cleavage, and developmental potential of cryopreserved oocytes related to the cumulus investment. In this, our results were similar to those of Whittingham (28) who found that the presence or absence of cumulus cells did not affect the fertilization rate nor the developmental ability of frozen-thawed mouse oocytes. Carroll et al. (3, 4) reported that the freezing-thawing of mouse oocytes reduced the fertilization rate, apparently by causing changes in the zona pellucida. By use of zona drilling, it was shown that the block to fertilization in frozen-thawed oocytes was at the level of the zona pellucida rather than at the vitelline membrane or in the cytoplasm. Our observations support this conclusion, inasmuch as large numbers of spermatozoa were bound firmly to the zona pellucida of frozen-thawed bovine oocytes and were very difficult to remove, even by washing 4x in culture medium or treatment with hyaluronidase, a feature that did not occur with unfrozen oocytes. The possibility of cryogenic genetic damage to oocytes, resulting in abnormal development of embryos and fetuses cannot be ruled out. Petzelt (20) has suggested that influences (such as freezing) which affect the spindle apparatus may impair chromosome segregation and produce aneuploid embryos. Indeed, Kola et al. (11) demonstrated a 3~ increase in the incidence of aneuploidy in mouse zygotes after either vitritication or slow freezing. Further research will no doubt address this question in the bovine. Meanwhile, this research has demonstrated that the production of calves from frozen-thawed oocytes, matured, fertilized, and cultured in vitro, is feasible. ACKNOWLEDGMENTS
We are sincerely grateful to Dr. T. Nagai, Dr. S. Abe, and Dr. S. Takahashi for their constructive ad-
OOCYTES
491
vice and to Ms. Y. Zenia and Ms. A. Akaike for the excellent technical assistance during the course of the experiments. REFERENCES
I, Bedford, J. M. Techniques and criteria used in the study of fertilization. In “Methods in Mammalian Embryology” (J. Daniel, Jr., Ed.), pp. 3763. Freeman, San Francisco, 1971. 2. Brackett, B., and Oliphant, G. Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. 12, 260-274 (1975). 3. Carroll, J., Depypere, H., and Matthews, C. D. Freeze-thaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozen-thawed mouse oocytes. J. Reprod. Ferfil. 90, 547-553 (1990). 4. Carroll, J., Warnes, G. M., and Matthews, C. D. Increase in digyny explains polyploidy after in vitro fertilization of frozen-thawed mouse oocytes. J. Reprod. Fertil. 85, 489-494 (1989). 5. Downs. S. M., Schroeder, A. C., and Eppig, J. J. The developmental capacity of mouse oocytes following maintenance of meiotic arrest in vifro. Gamete Res. 15, 305-316 (1986). 6. Goto, K., Kajihara, Y., Kosaka, S., Koba, M., Nakanishi, Y., and Ogawa, K. Pregnancies after co-culture of cumulus cells with bovine embryos derived from in vitro matured follicular oocytes. J. Reprod. Fertil. 83, 753-758 (1988). 7. Hernandez-Ledezma, J. J., and Wright, R. W., Jr. Deepfreezing of mouse one-cell embryos and oocytes using different cryoprotectants. Theriogenology 32, 735-743 (1989). 8. Heyman, Y.. Smorag, Z., Katska, L., Vincent, C., Garneir, V., and Cognie, Y. Influence of carbohydrates, cooling and rapid freezing on viability of bovine non-matured oocytes or l-cell fertilized eggs. Cryo Lett. 7, 170-183 (1986). 9. Jackowski, S., Leibo, S. P., and Mazur, P. Glycerol permeabilities of fertilized and unfertilized mouse ova. J. Exp. 2001. 212, 329-341 (1980). IO. Ko, Y., and Threlfall, W. R. The effect of l,2propanediol as a cryoprotectant on the freezing of mouse oocytes. Theriogenology 29, 987-995 (1988). Il. Kola, I., Kirby, C., Shaw, J., Davey, A., and Trounson, A. Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses. Teratology 38, 467-474 (1988). 12. Kono, T., Kwon, 0. Y., and Nakahara, T. Development of vitrified mouse oocytes after in vitro fertilization. Cryobiology 28, 5&54 (1991). 13. Leibo, S. P., McGrath, J. J., and Cravalho, E. G. Microscopic observations of intracellular ice formation in unfertilized mouse ova as a func-
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tion of cooling rate. Cryobiology 15, 257-271 (1978). 14. Lim, J. M., Fukui, Y., and Ono, H. The post-thaw developmental capacity of frozen bovine oocytes following in vitro maturation and fertilization. Theriogenology 35, 12251235 (1991). 15. Nakagata, N. High survival rate of unfertilized mouse oocytes after vitrification. J. Reprod. Fertil. 87, 479-483 (1989). 16. Nakagata, N. Survival of mouse embryos derived from in vitro fertilization after ultrarapid freezing and thawing. J. Mamm. Ova Rex. 6, 23-26 (1989). 17. Ohgoda, O., Niwa, K., Yuhara, M., Takahashi, S., and Kanoya, K. Variations in penetration rates in vitro of bovine follicular oocytes do not reflect conception rates after artificial insemination using frozen semen from different bulls. Theriogenology 29, 1375-1381 (1988). 18. Parkening, T. A., Tsunoda, Y., and Chang, M. C. Effects of various low temperature, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozenthawed mouse eggs. J. Exp. Zool. 197, 369-374
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19. Pellicer, A., Lightman, A., Parmer, T. G., Behrman, H. R., and De Cherney, A. H. Morphological and functional studies of immature rat oocyte-cumulus complexes after cryopreservation. Fertil. Steril. 50, 805-810 (1988). 20. Petzelt, C. Biochemistry of the mitotic spindle. Int. Rev. Cytol. 60, 53-92 (1979). 21. Rail, W. F., and Fahy, G. M. Ice-free cryopreservation of mouse embryos at - 196” C by vitrification. Nature 313, 573-575 (1985). 22. Sathananthan, A. H., Ng, S. C., Trounson, A. O., Bongso, A., Ratnam, S. S., Ho, J., Mok, H., and Lee, M. N. The effects of ultrarapid freezing on meiotic and mitotic spindles of
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mouse oocytes and embryos. Gamete Res. 21, 38.5-401(1988). Schroeder A. C., Champlin, A. K., Mobraaten, L. E., and Eppig, J. J. Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J. Reprod. Fertil. 89, 43-50 (1990). Shioya, Y., Kuwayama, M., Fukushima, M., Iwasaki, S., and Hanada, A. In vitro fertilization and cleavage capability of bovine follicular oocytes classified by cumulus cells and matured in vitro. Theriogenology 30, 489-496 (1988). Sirard, M. A., Parish, J. J., Ware, C. B., Leibfried-Rutledge, M. L., and First, N. L. The culture of bovine oocytes to obtain developmentally competent embryos. Biol. Reprod. 39, 546-552 (1988). Trounson, A. Preservation of human eggs and embryos. Fertil. Steril. 46, I-12 (1986). Tsunoda, Y., Parkening, T. A., and Chang, M. C. In vitro fertilization of mouse and hamster eggs after freezing and thawing. Experientia 32, 223224 (1976). Whittingham, D. G. Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at - 196” C. /. Reprod. Fertil. 49, 89-94 (1977). Whittingham, D. G., Leibo, S. P., and Mazur, P. Survival of mouse embryos frozen to - 196°C and - 129°C. Science 187, 411414 (1972). Wilmut, 1. The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. L(fe Sci. 11, 1071-1079 (1972). Xu, P. K., and Betteridge, J. K. Cryopreservation of bovine oocytes using propylene glycol:Preliminary results. Theriogenology 37, 324 (1992). [Abstract]
CRYOBIOLOGY
(1992)
In Vitro Fertilization
E. FUKU,”
T. KOJIMA,t
and Development Bovine Oocytes
of Frozen-Thawed
Y. SHIOYA,‘F G. J. MARCUS,+
AND
B. R. DOWNEY”,’
*Department of Animal Science, Macdonald Campus, McGill University, Ste. Anne de Be//ewe, Quebec, Canada H9X lC0; fDepartment of Animal Reproduction. National Institute of Animal Industry, Tsukuba-Norindanchi, P.O. Box 5, Ibaraki, 305, Japan; and SCentre for Food and Animal Research Agriculture Canada, Ottawa, Ontario, Canada KIA OC6 Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%). but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a cl 1992 Academic Preab. Inc. recipient heifer resulted in pregnancy and the birth of twin calves.
Success in the cryopreservation of em- new interest in the preservation of oocytes bryos was first achieved in the pioneering at low temperatures. Thus far, some success has been work of Whittingham ef al. (29), with the mouse, in the United States, and indepen- achieved with human oocytes and with dently by Wilmut (30) in Great Britain. The mouse oocytes. Trounson (26) found that protocols developed by these workers have vitrification resulted in the greatest oocyte served as the basis for cryopreservation of survival and subsequent development in the embryos of most species and have led to vitro in the human. In early studies several widespread exploitation of cryopreservainvestigators obtained normal young from tion as a routine practice in the embryo- IVF of ovulated mouse oocytes frozen transfer industry. Hitherto, procedures for slowly using DMSO, albeit with low cryothe cryopreservation of oocytes have not survival and fertilization rates (16, 25, 26). been developed to the same extent, partic- Recently, Kono et al. (12) and Nakagata ularly in the case of farm animals, due to (15, 16) reported that mouse oocytes also factors such as the generally lower yield of can be cryopreserved using vitrification viable embryos resulting from in vitro mat- and retain competence to develop into noruration (IVM), fertilization (IVF), and sub- mal offspring following IVF-IVC and emsequent culture (IVC) as well as a lack of bryo transfer. Ko and Threlfall (10) reeconomic incentives. However, the recent ported that mouse oocytes can be slowly rapid improvement of IVM-IVF-IVC tech- frozen and preserved at - 196°C using 1,2niques in the bovine (6, 25) has generated propanediol as a cryoprotectant. Hitherto, however, limited progress in the cryopreservation of oocytes of farm animals has Received December 10, 1991; accepted February been achieved (14, 31) and production of 24, 1992. offspring has not been reported. In the cur’ To whom reprint requests should he addressed. 485 001 I-2240/92 $5.00 Copyright 0 1992 by Academic Press. Lnc All rights of reproduction in any form reserved.
486
FUKU
ET AL.
oocytes into 0.25 ml French straws [I.M.V., L’Aigle, France]). The ends of the straws were heat-sealed and the straws placed horizontally in the ethanol bath of a programmable freezer (MPF-40, Tokyo MATERIALS AND METHODS Rikakikai, Japan) kept at - 8°C. After 2 min, ice formation was induced by touching Oocytes the straw with forceps cooled to - 196°C in The cumulus-oocyte complex classifica- liquid nitrogen. When seeding of all straws tion and IVM-IVF-IVC procedures used in was complete, the bath was held at -8°C these experiments were essentially the for a further 10 min and then cooled at same as described previously by Shioya et 0.3”Cmin. When the bath temperature al. (24). After collection from abattoir ova- reached -4O”C, the straws were plunged ries, bovine cumulus-oocyte complexes into liquid nitrogen. After storage for l-2 were classified according to their morpho- weeks, straws were rewarmed by holding in logical characteristics, as follows: air for 5 s and then immersing them in water Class A had a compact, dense cumulus at 22°C. The straws were cut and the concell layer; Class B had a compact but sparse tents placed for 5 min in a watch glass in cumulus cell layer (B,) or was partially de- M199a containing 1.O M propanediol and nuded (B,)-the latter were used for oocyte 0.5 M sucrose and then transferred into 0.5 evaluation, as described below; Class C M sucrose in M199a for another 5 min to oocytes were devoid of cumulus cells and dilute the cryoprotectant. were discarded. The medium used for vitrification was In the first experiment, bovine cumulus- DAP 213 (16), a mixture of 2.0 M dimethyl oocyte complexes, at the germinal vesicle sulfoxide (DMSO, Dojin Chemical Inc., (GV) stage, were cryopreserved directly. In Kumamoto, Japan), 1.0 M acetamide the second experiment, cumulus-oocyte (Sigma), and 3.0 M propanediol in M199a. complexes were cultured for 21-26 h in 100 Cumulus-oocyte complexes were imp.1droplets of maturation medium (25 mM mersed in DAP213 at room temperature, Hepes-buffered TCM- 199 with 10% calf se- loaded into 0.25-ml straws, and plunged dirum and antibiotics, see Ref. (24)) under rectly into liquid nitrogen, the whole procemineral oil (Squibb and Sons, Inc., Prince- dure taking less than 1 min. After storage ton, NJ) at 39°C in 5% CO, in air and then for l-2 weeks, the straws were warmed by frozen. holding in air for 5 s and then immersing them in water at 22°C. The contents were Freezing and Thawing immediately diluted with a single addition Two different procedures, slow freezing of 0.5 M sucrose in M199a for 5 min and and vitrification, applied to GV-stage and then expelled into M199a containing 0.5 M to in vitro matured oocytes were compared. sucrose in a watch glass and held for 5 min. In the slow freezing, the cumulus-oocyte In Vitro Fertilization complexes were immersed directly into 2.0 The basic medium (Medium BO) used for M 1,2-propanediol (Sigma Chemical Co., St. Louis, MO) in Hepes-buffered TCM- the treatment of spermatozoa and the fer199 (GIBCO, Grand Island, NY) supple- tilization of oocytes was that used by mented with 0.4% bovine serum albumin Brackett and Oliphant (2) for the fertiliza(BSA, crystallized, Sigma) (- M199a), at tion of rabbit eggs in vitro. Two or three ca. 25°C and equilibrated for 10 to 20 min 0.5-ml straws of frozen semen obtained (including the time required to load the from a single Japanese black bull were rent study, two different freezing methods (vitrification and slow freezing), applied to immature and in vitro matured bovine oocytes, were compared.
DEVELOPMENT
OF FROZEN-THAWED
thawed in water at 30°C. Spermatozoa were washed 2x by suspension and centrifugation at 500g for 5 min in Medium BO supplemented with 10 mM caffeine-sodium benzoate (Sigma) without BSA (Caff-BO). The pellet was resuspended in 0.5 to 1.Oml Caff-BO and the sperm concentration measured with a haemocytometer and adjusted to 107/ml. The suspension was then diluted 1:1 with Medium BO supplemented with 20 mg/ml crystallized BSA (BSA-BO) and 5 ~1 heparin/ml (Novo-Heparin 1000, 1000 U/ml Novo Industry A/S, Japan). Droplets (0.1 ml) of this sperm suspension (5 x lo6 sperm/ml) were incubated for 15 min at 39°C in 5% CO, in humid air. Thawed GV-stage cumulus-oocyte complexes, cultured for 21-26 h or complexes frozen after maturation culture, were transferred into sperm droplets for fertilization. In addition, unfrozen cumulus-oocyte complexes, matured in vitro, were placed in sperm droplets, as controls. Sperm and cumulus-oocyte complexes were incubated for 6 h at 39°C in 5% CO* in air. After washing 4~ in culture medium, the cumulus-oocyte complexes were transferred onto a proliferating monolayer of cumulus cells in 100 ~1 droplets of culture medium (25 mM Hepes-buffered TCM-199 supplemented with 10% calf serum and antibiotics, see Ref. (24)) under mineral oil at 39°C in 5% CO2 in air, for assessment of cleavage and development.
487
OOCYTES
stained with 1% orcein in 45% (v/v) aqueous acetic acid and examined at 1000X with a phase-contrast microscope (1, 17). Eggs containing both female and male pronuclei were considered to be fertilized and were deemed normal or polyspermic according to the number of pronuclei in the cytoplasm. Development to the two-cell stage was assessed 40 h after placing oocytes from class A or B cumulus complexes with sperm. The developing embryos were cultured for an additional 5 days to evaluate their capacity to develop into morulae or blastocysts. Four embryos (two morulae and two blastocysts) derived from oocytes slowly frozen after IVM were placed in Hepesbuffered TCM199 supplemented with 10% calf serum, loaded into an 0.25-ml straw, and deposited nonsurgically in the left uterine horn, ipsilateral to a corpus luteum, in a Holstein recipient at Day 7 of the cycle (Day 0 = onset of estrus). The progress of pregnancy was monitored periodically by ultrasonic scanning. Statistical
Analysis
Data from eight replicate experiments were pooled and the treatment effects were compared by x2 analysis. RESULTS
Survival
of Cryopreserved
Oocytes
In preliminary experiments, oocytes frozen in the presence of 2M propanediol at Cryosurvival of oocytes was assessed7 h O.l”C/min to -40°C or at O.S”C/min to after thawing, using a stereomicroscope to -80°C survived poorly and none develcount the numbers of normal and pycnotic- oped to the two-cell stage after IVF (data appearing oocytes (the latter were assumed not shown). Recovery and morphological to be degenerate). To assess fertilization, survival of oocytes cryopreserved by slow class B, oocyte complexes were retrieved freezing (0.3”/min) and by vitrification are from the culture dishes 19-20 h after insem- compared in Table 1. Nearly 90% (1537/ ination and cumulus cells and spermatozoa 1764) of all cryopreserved oocytes were reattached to the zona pellucida removed covered. The survival rates (calculated with 0.1% hyaluronidase (Sigma). The from results pooled for A and B classes of oocytes were then fixed in 1:3 acetic acid: cumulus investment) were greater (P < ethanol for 24-28 h at room temnerature. , 0.05) after slow freezing than after vitrificaA Oocyte Evaluation
488
FUKU ET AL. TABLE 1 Effect of Freezing of Bovine Oocytes on Their Recovery and Morphological Survival* after Freezing-Thawing
Group GV, vitrified GV, frozen slowly IVM, vitrified IVM, frozen slowly
Class of cumulus investment?
No. of oocytes frozen+
No. (%) of oocytes recovered
No. (%) with normal morphology*
A B A B A B A B
120 176 129 135 351 258 325 270
100 (83.3) 160 (90.9) 107 (82.9) 121 (89.6) 316 (90.0) 226 (87.6) 269 (82.8) 238 (88.1)
13 (13.0)” 25 (15.6)” 31 (29.0)b 39 (32.2)b 45 (14.2)” 31 (13.7)O 148 (55.0)’ 122 (51.3)
* Assessed 7 h after thawing. t Class A had a compact, dense cumulus cell layer; Class B had a compact but sparse cumulus cell layer. $ GV and IVM data pooled from 3 and 5 replicates, respectively. a,b,cWithin columns, values with different superscripts are different (P < 0.05).
tion both in GV-stage oocytes (30.7 vs 14.6%) and in oocytes matured in vitro (53.3 vs 14.0%). Fertilization of Cryopreserved Oocytes Fertilization data are shown in Table 2. Oocytes frozen slowly after IVM were less capable of being fertilized (5 1%) than unfrozen, control oocytes (76%), but were more capable than vitrified oocytes or oocytes frozen prior to maturation (P < 0.05).
Development Potential of Cryopreserved Oocytes Table 3 shows the proportions of oocytes that cleaved and that developed to the morula/blastocyst stage. The proportion of cryopreserved oocytes which developed into two-cell embryos was greatest among those that had been frozen slowly after IVM (16%)) but lower than among unfrozen controls (40%, P < 0.05). However, there was no difference between unfrozen oocytes and slowly frozen IVM oocytes in
TABLE 2 The Fertilization of Cryopreserved Bovine Oocytes No. of oocytes fertilizedt
Group
No. of oocytes*
Total (%)
Normal
Polyspermic
GV, vitrified GV, frozen slowly IVM, vitrified IVM, frozen slowly IVM, control, not frozen
26 22 43 51 91
2 (7.7)” 2 (9.1)” 5 (11.6)” 26 (51.O)b 69 (75.8)
0 1 2 12 48
2 I 3 14 21
* GV and IVM data pooled from 3 and 5 replicates, respectively. The values are the total numbers of oocytes with normal post-thaw morphology. t Oocytes with male and female pronuclei. Normal and polyspermic fertilization were indicated by the presence of 2 and >2 pronuclei, respectively. o.bx Within columns, values with different superscripts are different (P < 0.05).
DEVELOPMENT
OF FROZEN-THAWED
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TABLE 3 Development Capacity of Cryopreserved Bovine Oocytes in Vitro Class of cumulus investment*
Group
No. of oocytes fertilized?
No. (%) two-cell embryos
No. of morulae (M) and blastocyst (B) {% of two-cell embryos}
I (1.1)” 1 (0.7)” l(1.1)” 2 (1.9)” 14 (4.9) 9 (4.2) 35 (16.2)b 26 (15.7)b 35 (41.7)’ 52 (38.0)’
O{ 0%) O{ O%} O{ 0%) GV, frozen slowly O{ O%} IVM, vitrified O{ 0%) B O{ 0%) A IVM, frozen slowly 4 (2B, 2M) {11.4%}$ B 2 (IB, IM) {7.7%} A IVM, control, not frozen 5 (5B) { 14.3%) B 5 (5B) {9.6%} * Class A had a compact, dense cumulus cell layer; Class B had a compact but sparse cumulus cell layer. t GV and IVM data were pooled from 3 and 5 replicates, respectively. $ Nonsurgical transfer of 2 B and 2 M into a recipient heifer resulted in a confirmed pregnancy. rr~h~c Within columns, values with different superscripts are different (P < 0.05). A B A B A
GV, vitrified
the proportions that developed into morulae or blastocysts after IVF (11.5 vs 9.8%). The class of cumulus investment of the oocytes at the time of their harvest from the ovary had no discernible effect on the developmental potential of the cryopreserved oocytes. Two morulae and two blastocysts that had developed from IVM oocytes, slowly frozen, thawed and fertilized in vitro, all transferred into a single Holstein recipient, produced a pregnancy. On April 3, 1992, twin calves were born; the female and male weighed 32.7 and 51.2 kg, respectively Fig. 1). DISCUSSION
Recently, Hernandez-Ledezma and Wright (7) reported that the use of propanediol instead of glycerol or DMSO significantly improved survival and development of cryopreserved mouse oocytes to the two-cell stage. Nakagata (15), using the medium of Rall and Fahy (21), found that ca. 88% of vitrified mouse oocytes were morphologically normal, 80% of these developed to the two-cell stage and, following
94 148 90 106 286 215 216 166 84 137
transfer, resulted in a pregnancy rate of 46%. However, inasmuch as oocytes and embryos at various stages of development differ both physiologically and morphologically (8, 9, 13), freezing techniques developed for the one may not be suitable for the other. Thus, Heyman et al. (8) reported that very few bovine oocytes (6%) matured in vitro after rapid freezing and thawing. In the present study also, ca. 30% of oocytes frozen slowly at the GV stage survived (Table l), but fewer than 5% underwent germinal vesicle breakdown (GVBD) and polar body formation (data not shown). Moreover, only three (1.5%) of these cleaved after IVF and none developed into morulae or blastocysts (Table 3). These findings contrast with those of Schroeder et al. (23) who found that GV-stage mouse oocytes survived slow freezing to -80°C 95% underwent maturation in vitro as indicated by GVBD and polar body formation (90%), but, as in the present study, few cleaved and none developed into blastocysts. This suggests that immature bovine ova are more sensitive to cryoprocessing than GVstage mouse oocytes. Sathananthan et al. (22) found that, in
490
FUKU ET AL.
FIG. 1. Twin calves (Holstein x Japanese Black) born from frozen-thawed oocytes, matured, fertilized, and cultured in vitro to morulaeMastocysts.
mouse oocytes, spindle microtubule reorganization after GVBD is particularly sensitive to cold and is readily damaged by exposure to low temperatures, the damage becoming apparent only at the time of the first mitotic division. This explains the finding that few bovine oocytes matured after cryopreservation at the GV stage and even fewer cleaved after IVF. However, Downs et al. (5) and Schroeder et al. (23) found that when meiosis was arrested with isobutyl-3-methylxanthine before cryopreservation, development was significantly improved after freezing and thawing. Exploration of such an approach should form the basis of future studies and may result in sufficiently high survival to permit the routine use of GV-stage oocytes. The finding that the cleavage rate after IVF of mature oocytes frozen slowly was higher than that of other treatment groups agrees with the similar findings of Schroeder et al. (23). Moreover, the potential of two-cell embryos derived from frozen oocytes to develop into morulae or blastocysts was not different from that of
embryos from unfrozen control oocytes. In both the present study and that of Schroeder et af. (23), the dramatic change in freezability was associated with meiotic maturation in vivo or in vitro. Moreover, the damaging effects of freezing-thawing were apparent only up to the two-cell stage but, past this hurdle, did not affect further development to morulae or blastocysts. Although mouse and human oocytes have been cryopreserved successfully by vitrification (15, 26), in the present study, vitrification applied to either immature or in vitro matured bovine oocytes resulted in very poor survival and cleavage and no development into morulae or blastocysts. Consequently, slow freezing to -40°C at 0.3”Clmin appears to be the preferred procedure for cryopreservation of bovine oocytes. Nevertheless, the nature of the effects of vitrification on bovine oocytes should be investigated as this relatively new approach is simple and easily applied under field conditions. In contrast to the observations of Pellicer er al. (19) and Schroeder ec al. (23) that
DEVELOPMENT
OF
FROZEN-THAWED
greater survival was associated with more layers of cumulus cells in frozen-thawed rat and mouse oocytes, we found no differences in survival, cleavage, and developmental potential of cryopreserved oocytes related to the cumulus investment. In this, our results were similar to those of Whittingham (28) who found that the presence or absence of cumulus cells did not affect the fertilization rate nor the developmental ability of frozen-thawed mouse oocytes. Carroll et al. (3, 4) reported that the freezing-thawing of mouse oocytes reduced the fertilization rate, apparently by causing changes in the zona pellucida. By use of zona drilling, it was shown that the block to fertilization in frozen-thawed oocytes was at the level of the zona pellucida rather than at the vitelline membrane or in the cytoplasm. Our observations support this conclusion, inasmuch as large numbers of spermatozoa were bound firmly to the zona pellucida of frozen-thawed bovine oocytes and were very difficult to remove, even by washing 4x in culture medium or treatment with hyaluronidase, a feature that did not occur with unfrozen oocytes. The possibility of cryogenic genetic damage to oocytes, resulting in abnormal development of embryos and fetuses cannot be ruled out. Petzelt (20) has suggested that influences (such as freezing) which affect the spindle apparatus may impair chromosome segregation and produce aneuploid embryos. Indeed, Kola et al. (11) demonstrated a 3~ increase in the incidence of aneuploidy in mouse zygotes after either vitritication or slow freezing. Further research will no doubt address this question in the bovine. Meanwhile, this research has demonstrated that the production of calves from frozen-thawed oocytes, matured, fertilized, and cultured in vitro, is feasible. ACKNOWLEDGMENTS
We are sincerely grateful to Dr. T. Nagai, Dr. S. Abe, and Dr. S. Takahashi for their constructive ad-
OOCYTES
491
vice and to Ms. Y. Zenia and Ms. A. Akaike for the excellent technical assistance during the course of the experiments. REFERENCES
I, Bedford, J. M. Techniques and criteria used in the study of fertilization. In “Methods in Mammalian Embryology” (J. Daniel, Jr., Ed.), pp. 3763. Freeman, San Francisco, 1971. 2. Brackett, B., and Oliphant, G. Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. 12, 260-274 (1975). 3. Carroll, J., Depypere, H., and Matthews, C. D. Freeze-thaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozen-thawed mouse oocytes. J. Reprod. Ferfil. 90, 547-553 (1990). 4. Carroll, J., Warnes, G. M., and Matthews, C. D. Increase in digyny explains polyploidy after in vitro fertilization of frozen-thawed mouse oocytes. J. Reprod. Fertil. 85, 489-494 (1989). 5. Downs. S. M., Schroeder, A. C., and Eppig, J. J. The developmental capacity of mouse oocytes following maintenance of meiotic arrest in vifro. Gamete Res. 15, 305-316 (1986). 6. Goto, K., Kajihara, Y., Kosaka, S., Koba, M., Nakanishi, Y., and Ogawa, K. Pregnancies after co-culture of cumulus cells with bovine embryos derived from in vitro matured follicular oocytes. J. Reprod. Fertil. 83, 753-758 (1988). 7. Hernandez-Ledezma, J. J., and Wright, R. W., Jr. Deepfreezing of mouse one-cell embryos and oocytes using different cryoprotectants. Theriogenology 32, 735-743 (1989). 8. Heyman, Y.. Smorag, Z., Katska, L., Vincent, C., Garneir, V., and Cognie, Y. Influence of carbohydrates, cooling and rapid freezing on viability of bovine non-matured oocytes or l-cell fertilized eggs. Cryo Lett. 7, 170-183 (1986). 9. Jackowski, S., Leibo, S. P., and Mazur, P. Glycerol permeabilities of fertilized and unfertilized mouse ova. J. Exp. 2001. 212, 329-341 (1980). IO. Ko, Y., and Threlfall, W. R. The effect of l,2propanediol as a cryoprotectant on the freezing of mouse oocytes. Theriogenology 29, 987-995 (1988). Il. Kola, I., Kirby, C., Shaw, J., Davey, A., and Trounson, A. Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses. Teratology 38, 467-474 (1988). 12. Kono, T., Kwon, 0. Y., and Nakahara, T. Development of vitrified mouse oocytes after in vitro fertilization. Cryobiology 28, 5&54 (1991). 13. Leibo, S. P., McGrath, J. J., and Cravalho, E. G. Microscopic observations of intracellular ice formation in unfertilized mouse ova as a func-
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tion of cooling rate. Cryobiology 15, 257-271 (1978). 14. Lim, J. M., Fukui, Y., and Ono, H. The post-thaw developmental capacity of frozen bovine oocytes following in vitro maturation and fertilization. Theriogenology 35, 12251235 (1991). 15. Nakagata, N. High survival rate of unfertilized mouse oocytes after vitrification. J. Reprod. Fertil. 87, 479-483 (1989). 16. Nakagata, N. Survival of mouse embryos derived from in vitro fertilization after ultrarapid freezing and thawing. J. Mamm. Ova Rex. 6, 23-26 (1989). 17. Ohgoda, O., Niwa, K., Yuhara, M., Takahashi, S., and Kanoya, K. Variations in penetration rates in vitro of bovine follicular oocytes do not reflect conception rates after artificial insemination using frozen semen from different bulls. Theriogenology 29, 1375-1381 (1988). 18. Parkening, T. A., Tsunoda, Y., and Chang, M. C. Effects of various low temperature, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozenthawed mouse eggs. J. Exp. Zool. 197, 369-374
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