Effect of rapid addition and dilution of dimethyl sulfoxide on the viability of frozen-thawed rabbit morulae

The aim of the present study was to examine effects of altering thawing conditions and procedure of addition and dilution of Me2SO on the viability of frozen-thawed rabbit morulae. Five hundred and sixty two rabbit morulae were cooled from room temperature to -80 degrees C at 1 degree C/min in the presence of 1.5 M dimethyl sulfoxide (Me2SO) using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, cooled rapidly, and stored in liquid nitrogen. When Me2SO was added in a single step, the frozen embryos were thawed in ambient air at 40 degrees C/min and Me2SO was diluted in a single step, 99 of 107 (93%) embryos cultured for 48 hr and 12 of 32 (38%) embryos transferred to 6 recipients developed to expanding blastocysts and viable fetuses, respectively. When Me2SO was added in a single step and the frozen embryos were thawed at the same rate and transferred directly without removal of Me2SO to culture media or oviducts of 8 recipients, 67 of 75 (89%) embryos cultured and 12 of 40 (30%) embryos transferred developed to expanding blastocysts and viable fetuses, respectively. There were no significant differences between these survival rates and survival rates obtained by conventional method, i.e., frozen embryos were thawed at 4 degrees C/min by interrupted slow method and Me2SO was added and diluted in a stepwise manner.     

Survival of rabbit embryos after culture or culture/freezing

Five-hundred-and-ninety-five rabbit 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 −196°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 frozen-thawed embryos were transferred into pseudopregnant recipients in an earlier physiological stage (−24 h).     

The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.     

Survival of 16-celled and morula stage rabbit embryos frozen to -196 degrees C,.

Preimplantation stage (16-celled and morula) rabbit embryos were successfully frozen to -196 degrees C. The cooling rate (from a room temperature to 0 degrees C), the presence of the mucin layer surrounding embryos, the ice-seeding treatment and the thawing procedure were examined to determine their effects on the survival of the frozen embryos of Japanese white, New Zealand white and Dutch-Belted rabbits. A high proportion (51%; 16-celled, 69%; morula) of Dutch-Belted rabbit embryos developed in vitro, when they were frozen to -196 degrees C, applying the ice-seeding at -4 degrees C in the presence of 12.5% DMSO, after being cooled to 0 degrees C at the rate of 7-9 degrees C/min, and were diluted by a stepwise addition of 4 different strength PBS on thawing. The highest rate of in vitro development (81%; Japanese white, 75%; New Zealand white, 82%; Dutch Belted embryos) was obtained when the morula stage embryos were frozen to -196 degrees C applying seeding at -4 degrees C after being cooled to 0 degrees C at the rate of 1 degrees C/2.5 min and were diluted, on thawing, by stepwise addition of 6, 3 and 1% DMSO solution and a culture medium. No great difference was found in the survival rate between the embryos covered with the mucin layer and those which had not the coat. All the embryos frozen without applying seeding treatment failed to develop in vitro after being thawed and diluted. Nine out of 27 does each of which received 6 reimplantations of the embryos frozen-thawed became pregnant and were found to be carrying 37 normal fetuses on the 12th day of pregnancy.     

Conventional slow freezing, vitrification and open pulled straw (OPS) vitrification of rabbit embryos

Three different methods of cryopreservation viz., conventional slow freezing, vitrification and open pulled straw vitrification were compared for their ability to support post thaw in vitro and in vivo development of rabbit embryos. Morula stage rabbit embryos were collected from super-ovulated donor does. They were randomly allocated to different freezing methods and stored up to 3 months in liquid nitrogen. After thawing and removal of cryoprotectants, embryos exhibiting intact zona pellucida and uniform blastomeres were considered suitable for in vitro culture and/or transfer. Three to five cryopreserved embryos placed in approximately 1 ml of culture medium (TCM 199 supplemented with foetal calf serum and antibiotics) were incubated for up to 72 h under humidified atmosphere of 5% CO2 in air at 39 degrees C. Development to hatched blastocyst stage was considered the initial indicator of success of cryopreservation of embryos. Of the embryos cryopreserved by programmed freezing, open pulled straw vitrification, vitrification-55 h pc and vitrification-72 h pc 55, 71, 17 and 48%, respectively, developed into hatched blastocysts. Similarly 19, 29, and 4% of embryos cryopreserved by programmed freezing, open pulled straw vitrification and vitrification -72 h pc developed into live offspring on transfer to recipient does. This is the first report on open pulled straw vitrification of rabbit embryos. Present results, suggest that (a) open pulled straw vitrification supports better in vitro survival of frozen thawed rabbit morulae; (b) both programmed freezing and OPS are similar but superior to vitirification in supporting in vivo survival of frozen thawed rabbit embryos.     

Production of transgenic mice following deoxyribonucleic acid microinjection and embryo freezing

Experiments with mouse embryos were designed to assess the feasibility of freezing embryos after DNA microinjection. One-cell pronuclear stage mouse embryos were microinjected with cloned deoxyribonucleic acid (DNA) and cultured in vitro to the late eight-cell stage. Microinjected and matched control embryos were frozen and stored in liquid nitrogen. Following thawing, embryos were cultured for 8 h and transferred to recipient females. In a separate set of experiments, embryos were transferred to recipients immediately following DNA microinjection. Control (uninjected) embryos developed to the late eight-cell stage significantly better than surviving microinjected embryos. Of the embryos thawed, 76% of the microinjected and 60% of the control embryos survived to be transferred to recipients. Progeny were obtained with similar survival rates from both groups following embryo transfer with transgenic mice identified among the progeny from microinjected embryos. Mouse embryos can be microinjected with DNA, cultured in vitro, frozen, thawed, transferred to recipients and transgenic progeny can be obtained.     

Tolerance to vitrification of rat embryos at various developmental stages

Numerous genetically engineered rat strains have been produced via genome editing. Although freezing of embryos is helpful for the production and storage of these valuable strains, the tolerance to freezing of embryos varies at each developmental stage of the embryo. This study examined the tolerance to freezing of rat embryos at various developmental stages, particularly at the pronuclear stage. Embryos that had developed to the pronuclear, 2-cell, and morula stages were frozen via vitrification using ethylene glycol- and propylene glycol-based solutions. More than 90% of the embryos at all developmental stages survived after warming. The developmental rates to offspring of thawed embryos at the pronuclear, 2-cell, and morula stages were 19%, 41%, and 52%, respectively. Pronuclear stage embryos between the early and late developmental stages were then vitrified. The developmental rates to offspring of the thawed pronuclear stage embryos collected at 24, 28, and 31 h after the induction of ovulation were 17%, 21%, and 23%, respectively. These results indicated that the tolerance to vitrification of rat embryos increased with the development of embryos. The establishment of vitrification method of rat embryos at various developmental stages is helpful for improving the production and storage of valuable rat strains used for biomedical science.     

Some factors affecting the viability of mouse and cattle embryos frozen to −40°C before transfer to liquid nitrogen

A total of 1161 8- to 16-cell mouse embryos and 31 cattle early morulae and late blastocysts were frozen to ?40°C before transfer to liquid nitrogen. After thawing, mouse embryo viability was determined by in vitro development to the blastocyst stage and cattle embryo viability by both in vivo and in vitro development.Using glycerol as the cryoprotective agent, 88% of the mouse embryos developed to the blastocyst stage: thawing at 45 and 360° C/min gave the best results (88.8 and 84.8%, respectively). In another test with holding times at ?40°C of up to 60 min, about 70% of embryos developed to blastocysts with holding time 30–60 min.In cattle, 11 embryos frozen in DMSO and thawed at 360°C/min were transplanted to eight recipients. Four pregnancies (six fetuses) resulted. Thawing rates of 200 and 360°C/min resulted in the best in vitro development of cattle embryos.     

Liquid nitrogen vapour freezing of mouse embryos

Mouse morulae were frozen rapidly to -196 degrees C in the presence of glycerol by a two-step procedure; the embryos were transferred directly from -7 degrees C after seeding into liquid nitrogen vapour at -170 to -180 degrees C and then into liquid nitrogen 10-15 min later. Suitable conditions for the survival of embryos frozen with liquid nitrogen vapour were found to be: 2 M-glycerol, 2 M-propylene glycol, 2 M-ethylene glycol; 5-30 min equilibration time at 0 degrees C; 3-60 min holding time in liquid nitrogen vapour; dilution of glycerol with sucrose out of the frozen-thawed embryos; morula and early blastocyst stage embryos. Relatively high survival rates (69-74%) were obtained after rapid freezing by liquid nitrogen vapour. Morulae frozen in this fashion, cultured and transferred to recipients developed into normal young.     

Deep freezing of sheep embryos.

Sheep embryos, collected 1-8 days after oestrus, were placed in Dulbecco's phosphate-buffered saline medium (PBS). After treatment, the viability of the embryos was tested by temporary transfer to ligated rabbit oviducts. In Exp. 1, Days 5-8 embryos survived for at least 15 min at 0 degrees C in the presence of 1-5 M-DMSO. In Exp. 2, 12/14 Days 5-8 embryos survived after being frozen in 1-5 M-DMSO at 0-3 degrees C/min to temperatures ranging between-15 degrees and -60 degrees C and then thawed at 12 degrees C/min. In Exp. 3, Days 5-8 embryos were frozen in 1-5 M-DMSO at 0-3 degrees C/min to below-65 degrees C before being transferred to liquid nitrogen (-196 degrees C), and stored for 12 hr to 1 month. The embryos were thawed at 3 degrees C/min, 12 degrees C/MIN or 360 degrees C/min and, after transfer to rabbit oviducts, 0/4, 10/36 and 1/4, respectively, developed normally. The 11 embryos which were considered normal when recovered from the rabbit oviducts plus 1 slightly retarded embryo were transferred to 7 recipient ewes. Four ewes subsequently lambed, producing 5 lambs. In addition, 8 embryos were transferred to 4 ewes directly after thawing. Three of these ewes subsequently lambed, producing 3 lambs.     

Developmental capacity of bovine oocytes cryopreserved after maturation in vitro and of frozen-thawed bovine embryos derived from frozen mature oocytes

The present study was conducted 1) to investigate the post-thaw developmental capacity of in vitro mature bovine oocytes (Metaphase II) frozen by 1.6 M of 1,2-propanediol and 2) to confirm the viability of frozen bovine embryos derived from frozen mature oocytes. The cleavage and developmental rates to the blastocyst stage of frozen-thawed mature oocytes were significantly lower (P<0.01) than that of nonfrozen oocytes. When mature oocytes were treated with hyaluronidase, trypsin, or base solution (solution control) before processing to remove the cumulus cells, the developmental rates to the blastocyst stage of frozen-thawed oocytes were 2.8% (5 180 ), 3.1% (9 295 ) and 1.1% (1 89 ), respectively. The viability and developmental capacity of frozen-thawed bovine embryos derived from frozen mature oocytes were not different from those of frozen-thawed bovine embryos derived from nonfrozen mature oocytes (control). Furthermore, nonfrozen and frozen-thawed embryos derived from frozen-thawed mature oocytes were nonsurgically transferred to recipient cows. One of the four and one of the two recipient cows became pregnant, respectively. The results of this study demonstrated the viability of embryos obtained from frozen-thawed bovine oocytes at Metaphase II followed by in vitro fertilization and culture to the blastocyst stage in vitro.     

小鼠不同阶段胚胎玻璃化冷冻保存的比较研究

采用玻璃化冷冻法对ICR、C57BL/6、DBA~*C57BL/6杂交F1代三种品系小鼠的不同阶段胚胎进行冷冻保存,比较胚胎解冻后形态良好率、体外发育率和移植后的出生率,结果表明解冻后各品系小鼠胚胎从2细胞到桑椹胚形态良好率在75%以上,其中8细胞胚胎形态良好率在83%以上,而囊胚的形态良好率仅在40%左右。解冻后胚胎体外培养的发育率随胚胎发育阶段的提高而提高,桑椹胚的发育达93%以上。体外受精2细胞冷冻胚与体内受精2细胞冷冻胚比较,二者形态良好率差异无显著意义(74%∶75%),但体内受精冷冻胚的发育率明显高于体外受精冷冻胚(76%:40%,p<0.01);胚胎经过三次反复冻融后形态良好率无显著差别;冷冻2细胞胚移植后的受孕率与仔鼠出生率分别达64%和40%,但均低于新鲜2细胞胚。     

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.     

Survival of frozen-thawed sheep embryos cryopreserved at cleavage stages

This study evaluated the effect of freezing-thawing procedures on the viability of sheep embryos cryopreserved at various developmental stages. The survival rates of frozen-thawed embryos were compared with non-frozen counterparts. Embryos were recovered from the oviduct and uterus, at different days of the early luteal phase, and were classified at six different developmental stages: 2- to 4-cell (n = 72), 5- to 8-cell (n = 73), 9- to 12-cell (n = 70), early morulae (n = 42), morulae (n = 41), and blastocyst (n = 70). For each early cleavage stage and blastocysts, approximately half of the embryos, were frozen immediately by slow freezing with an ethylene glycol-based solution. The remaining embryos were cultured to the hatched blastocyst stage. All morulae and compact morulae were frozen after recovery with the same protocol. Cryoprotectants were removed using 1M sucrose solution, and then warmed the embryos were cultured to the hatched stage in a standardized in vitro culture. Embryo developmental stage had a significant effect on the ability to hatch following freezing (P<0.0001). The cryotolerance of the embryos fitted a regression (r2 = 0.908), increasing linearly from 2- to 4-cell embryos (17.1%) to morula stage (46.3%) and in a quadratic regression from the morula to the blastocyst stage (83.7%). Frozen early cleavage stage embryos had a significantly lower viability than their fresh counterparts (23.1 vs 83.1%; P<0.0001), with a similar rate of viability between fresh or frozen blastocysts (92.5 vs 83.7%). In conclusion, early sheep embryos are very sensitive to freezing per se and the survival rates following conventional freezing improve as embryo developmental stage progresses.     

In vitro development and post-thaw survival of blastocysts derived from delipidated zygotes from domestic cats

The ability to cryopreserve in vitro-produced feline embryos was investigated. To improve the survival rate of cryopreserved embryos, first the developmental ability of in vitro fertilized feline zygotes (after removal of intracellular lipids) was determined, followed by the post-thaw survival of cryopreserved blastocysts derived from delipidated zygotes. More than 67% of the delipidated zygotes cleaved and 36% of them developed to the morula stage. The developmental ability of delipidated zygotes to the blastocyst stage (26%) was similar to that of sham-operated (30.5%) or control embryos (31.3%). Although the survival rate of delipidated blastocysts (81.8%) after freezing and thawing tended to be higher than that of control embryos without delipidation (60.6%), rates were not significantly different between the both groups. In conclusion, in vitro-produced feline blastocysts were successfully frozen, removal of the cytoplasmic lipid content in feline zygotes did not impair their in vitro developmental competence (up to the blastocyst stage), and reduction of cytoplasmic lipids by aspiration had no apparent effects on the survival of in vitro-derived blastocysts after cryopreservation.     

小鼠不同阶段胚胎玻璃化冷冻保存的比较研究

The cryopreservation of different embryo stages collected from ICR, C57BL/6 and F1 of DBA*C57BL/6 was carried out by using vitrification method. The morphology, in vitro development and birth rates of these embryos were compared after frozen-thawed. The results showed that more than 75% of the morphology from 2-cell embryos to morula stages from different strains was normal, the normal morphology rates of 8-cell embryos being the highest, while those of blastulas being the lowest. The in vitro development rates became higher as the embryos developed. The morphology of in vivo and in vitro fertilized frozen 2-cell embryos showed no difference, but the development rate of in vivo fertilized frozen 2-cell embryos was significantly higher than that of in vitro ones. Embryos that underwent 3 times frozen-thawing remained normal morphology. The pregnant rate and birth rate of frozen 2-cell embryos after embryo transfer were 64% and 40% respectively, but lower than those of fresh 2-cell embryo transfer.     

Effect of rapid addition and dilution of dimethyl sulfoxide and 37 degrees C equilibration on viability of rabbit morulae thawed rapidly

The aim of the present study was to examine the effects of various conditions of addition and dilution of dimethyl sulfoxide (Me2SO) and 37 degrees C equilibration, and also the effects of freezing in the solution which was prepared in advance and stored in plastic straws at -20 degrees C on the viability of rabbit morulae thawed rapidly. The embryos were cooled from room temperature to -30 degrees C at 1 degree C/min in the presence of 1.5 M Me2SO using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, then cooled rapidly, and stored in liquid nitrogen. The frozen straws were thawed rapidly (greater than 1000 degrees C/min). When Me2SO was added in a single step, equilibrated with embryos at 37 degrees C for 15 min and diluted out in a single step, a very high survival was obtained: transferable/recovered, 90%: developed/recovered, 96%. When embryos were pipetted into 1.5 M Me2SO that was prepared in advance, stocked in straws at -20 degrees C, and cooled, the proportions of transferable and developed embryos were equivalent to those of embryos frozen in the solution that was prepared immediately before use.     

Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro

The survival and developmental capacity of cumulus cell-enclosed oocytes frozen (1) at the germinal vesicle (GV) stage, after maturation in vitro with (2) and without (3) FSH, and (4) after gonadotrophin-stimulated ovulation were assessed. Survival, defined as the number of morphologically normal oocytes, after freeze-thaw at the GV stage (69%), was lower than for oocytes frozen after ovulation (84%), and after maturation in vitro with FSH (88%) and without FSH (81%). Treatment with DMSO without freezing had no effect on survival when compared with untreated controls except in immature GV-stage oocytes for which there was a slight reduction. After insemination in vitro, 9% of frozen-thawed GV-stage oocytes cleaved to two equal blastomeres, but none developed to blastocysts. Of oocytes matured in vitro before freezing, 17% cleaved to the 2-cell stage and 18% of these developed to blastocysts. When oocytes were matured in vitro in the presence of FSH, however, the percentage cleaving to the 2-cell stage after freeze-thaw was improved to 55%, and 77% of 2-cell stage embryos developed to blastocysts. When ovulated cumulus cell-enclosed oocytes were frozen, 88% cleaved and 67% of the cleaved embryos developed to blastocysts. When 158 two-cell embryos resulting from oocytes matured in vitro with FSH were transferred to the oviducts of pseudopregnant foster mothers, 41 genetically marked live young were produced (26%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Osmotic and cryoprotective effects of a mixture of DMSO and ethylene glycol on rabbit morulae

Comparisons were made of the osmotic and cryoprotective effects on rabbit embryos preserved by vitrification with 2 solutions and by conventional freezing. Embryos obtained from rabbits killed 70 to 72 h after mating were used in the study (n = 948). Initially, toxicity of the 3 cryoprotectants was studied in fresh (unfrozen) embryos (n = 135). Subsequently, embryos placed in ethylene glycol (EG, 40% v/v; n = 88) and ethylene glycol with dimethyl sulfoxide (EG+DMSO, 20% v/v each, respectively; n = 344) were loaded into straws and plunged directly into liquid nitrogen. Embryos placed in 1.5 M DMSO and 20% heat inactivated rabbit serum were subjected to conventional freezing in a programmable freezer (control group, n = 363). The osmotic effect was estimated by measuring the changes in the embryonic and interzonal volume (crossectional area) and in the thickness of the mucin coat (n = 18). Cryoprotective effectivity was determined by development to the blastocyst stage in vitro, or birth of normal pups after transfer into synchronized recipients. Osmotic effects of cryoprotective solutions on embryonic and interzonal volume and mucin coat thickness were variable and overall not significant. Survival rate of cryopreserved embryos in vitro and development to blastocysts, was worst in the EG-treated embryos. Survival rate at birth was higher in vitrified vs frozen embryos. We conclude that rabbit morulae can be vitrified successfully in EG+DMSO medium.     

Quick freezing of one-cell mouse embryos using ethylene glycol with sucrose

One-cell mouse embryos were frozen by direct plunging into liquid nitrogen (LN(2)) vapor after equilibration in 3 M ethylene glycol with 0.25 M sucrose (freezing medium) for 5 to 40 minutes. After thawing, the embryos were cultured in vitro and the effects of the equilibration period and dilution method were examined. No significant difference was observed in the in vitro survival of embryos when 0.5 or 1.0 M sucrose was used for the dilution of the cryoprotectant for each equilibration period. The highest survival rate (67.2%) was obtained when the embryos were equilibrated for 10 minutes, and the cryoprotectant diluted with either 0.5 or 1.0 M sucrose after thawing. Shorter (5 minutes) or prolonged (40 minutes) equilibration of embryos in the freezing medium yielded significantly lower survival rates. Dilution by direct transfer of the frozen-thawed embryos into PB1 resulted in lower survival rates than when 0.5 or 1.0 M sucrose was used. The in vitro development to the blastocyst stage of one-cell mouse embryos frozen after 10 minutes equilibration in the freezing medium and diluted after thawing in 0.5 M sucrose was significantly lower than the control (68.0 vs 92.7%). However, transfer of the blastocysts developing from frozen-thawed one-cell mouse embryos into the uterine horns of the recipients resulted in fetal development and implantation rates similar to the control.