Fast freezing of cow embryos in French straws with an automatic program

Cow embryos between day 6.5 and 9 were frozen in 1.5M DMSO in PBS at 2 degrees C/min from seeding to -25 degrees C before being plunged into liquid nitrogen directly or after 10 min at -25 degrees C. Cooling rate from 20 degrees C to -5 degrees C was 9 degrees C/min. Seeding was induced automatically at -5 degrees C by injection of liquid nitrogen vapour. Embryos were subsequently thawed by direct transfer to water at 20 degrees C (group I) or at 37 degrees C (group II). Survival was assessed by culture in vitro and by transfer. In group I, 35.7% were degenerated after thawing (compared to 35.4% in group II). Survival rate after culture in vitro for 24h was not significantly different (48.3% vs 42.8%) and hatching rate after 96h culture was quite similar (33.3% vs 34.4%). In group II, four pregnancies were obtained from 10 embryos transferred. Time at -25 degrees C did not improve the results. Automatic seeding did not impair survival. These results show that the quality of the embryo is the determinant factor for survival after freezing and that the plastic straw is the most suitable vessel for freezing, storage and transfer of embryos.     

A new procedure for the cryopreservation of equine embryos

Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2x2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN(2)) at -33 C or -38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to -6 C and then seeded, 0.3 C/min to -30 or -35 C and 0.1 C/min to -33 or -38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Ham's F10 with 5% FCS. The mean post-thaw quality of embryos plunged at -33 C was superior (P<0.05) to that of embryos plunged at -38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at -38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at -33 C or frozen in straws and plunged at -33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at -33 C resulted in higher quality embryos than plunging at -38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at -33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate (8 10 , 80%) than expanded blastocysts (1 7 , 14%).     

Observations on in vitro development of bovine morulae in Ham's F-10 and Dulbecco's phosphate buffered saline supplemented with normal steer serum

This study was conducted to compare in vitro development of bovine morulae in Ham's F-10 and Dulbecco's phosphate buffered saline (D-PBS) media supplemented with 10% (v/v) normal steer serum. Fifty-three excellent and good embryos were obtained by superovulating 15 non-lactating Holstein cows. Embryos were placed randomly in culture with Ham's F-10 or D-PBS media and development was recorded at 12-h intervals for the duration of culture. All embryos reached early blastocyst, blastocyst and expanded blastocyst stage. Nineteen of 27 embryos (70.1%) cultured in Ham's F-10 developed to hatched blastocyst stage in contrast to three out of 26 in D-PBS (11.5%). The mean developmental scores at 24, 48, 72, 96 and 120 h of culture were significantly (P<0.001) higher for embryos cultured in Ham's F-10. Also, the mean times to reach early blastocyst (25.84 +/- 6.65 vs 46.67 +/- 9.99 h), blastocyst (44.57 +/- 11.45 vs 61.89 +/- 16.62 h) and expanded blastocyst stage (65.00 +/- 13.20 vs 73.41 +/- 15.80 h) were significantly (P<0.001) shorter for embryos cultured in Ham's F-10. No difference was observed in the mean time to reach hatching (90.00 +/- 10.85 vs 84.00 +/- 16.97 h) and hatched blastocyst stage (97.26 +/- 18.71 vs 96.00 +/- 0.00 h). The results obtained support the concept that Ham's F-10 and normal steer serum provide for optimal bovine embryo development and suggest that 10% normal steer serum could be used as a protein supplement with D-PBS for short term storage and culture of bovine embryos.     

Use of sucrose during removal of cryoprotectants after thawing eight-cell mouse embryos

Eight-cell mouse embryos were frozen in 0.5-ml plastic straws in modified Dulbecco's phosphate buffered saline (PBS) plus 5% steer serum plus either 1.32 M dimethyl sulfoxide (DMSO) or 1.32 M glycerol. Upon thawing, embryos were diluted 1:4 with 0.0, 0.2, 0.6, or 1.0 M sucrose solutions within the straws. Thawing was either in air at ambient temperature or in 8 degrees C or 38 degrees C water. After 48 h of culture, more embryos frozen in DMSO and thawed in 8 degrees C and 37 degrees C water developed to blastocysts (87 and 93%, respectively) than embryos thawed in air (75%; P < 0.05). No significant differences in development were noted among the three thawing regimens when embryos were frozen with glycerol. There was no significant effect of concentration of sucrose during dilution on development of embryos postthaw. With glycerol as the cryoprotectant, damage to zonae pellucidae increased as thawing rates increased, whereas the opposite was observed with DMSO as the cryoprotectant (P < 0.05).     

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.     

Two step freezing of cow embryos in 1.4 M glycerol

Day 6 1 2 -7 1 2 cow embryos were frozen in 1.4 M glycerol in PBS, at 0.3 degrees C/min to -30 (group I), -35 (group II), and -40 degrees C (group III) before being plunged into liquid nitrogen. They were subsequently thawed by direct transfer to water at 37 degrees C. In Experiment I, embryos frozen and thawed were cultured in vitro, 12 out of 19 embryos (63%) survived and there were no significant (p > 0.05) differences in survival rates among the three freezing groups. In Experiment II, 29 embryos frozen to -30 or -35 degrees C were transferred non-surgically to heifers on day 7. Seventeen of 29 recipients (59%) were pregnant at day 60. Five embryos frozen to -35 degrees C resulted in 5 pregnancies (100%) after thawing and surgical transfer.     

In vitro survival of fresh and frozen/thawed bovine demi-embryos

Three experiments were conducted to investigate the effects of type of culture medium in freshly bisected bovine embryos and the effects of agar embedding and of 1.2 propanediol (PROH) as the cryoprotectant in frozen/thawed bisected bovine embryos on development in vitro. A total of 265 bovine embryos were used as controls or were microsurgically bisected and were cultured in vitro for 48 hours and development was determined 24 and 48 hours after the onset of culture. Whitten's medium supported more (P<0.05) intact and demi-embryos to grow to expanded blastocysts (92.9 and 73.1%, respectively) compared with Ham's F10 (43.8 and 26.3%, respectively) and PBS (53.8 and 12.5%, respectively). Embedding in agar and culture in Whitten's medium resulted in a higher (P<0.05) percentage of in vitro development of frozen/thawed demi-embryos after 24 hours than the freezing of nonembedded demi-embryos (44.1 versus 19.6%, respectively). This difference disappeared, however, after a 48 hours culture period (17.6 versus 11.8%, respectively). Following freezing in PROH, survival rates of 40 and 28%, respectively after 24 hours of culture were obtained for intact and demi-embryos. The respective percentages after 48 hours were 8.6 and 16%. Since neither embedding in agar nor the use of PROH as the cryoprotectant resulted in high survival rates of frozen/thawed demi-embryos in vitro, new freezing procedures are needed to overcome the sensitivity of demi-embryos to freezing and thawing.     

The viability of deep-frozen cow embryos

Day 7 cow embryos were frozen in 1.5 M-DMSO in PBS at 0.3 degrees C/min to -36 degrees C and at 0.1 degrees C/min between -36 and -60 degrees C before being plunged directly into liquid nitrogen. They were subsequently thawed (rapidly to -50 degrees C, at 4 degrees C/min from -50 to -10 degrees C, and rapidly again) to room temperature. Embryonic viability was tested by four different transfer techniques. Maximum pregnancy rate (8/12) was obtained with surgical transfer immediately after thawing and dilution of DMSO.     

Cryopreservation of murine embryos with trehalose and glycerol

Several concentrations of trehalose (0.0, 0.04, 0.1, 0.25 M) in combination with three concentrations of glycerol (1.0, 1.5, 2.0 M) were evaluated for the cryopreservation of murine embryos. Embryos were transferred through increasing concentrations of glycerol in Dulbecco's phosphate-buffered saline with 10% fetal calf serum (PBS + FCS) to reach the final glycerol concentrations. They were then randomly assigned to one of the concentrations of trehalose. A total of 506 morulae were packaged individually in 0.25-ml plastic straws and cooled from ambient temperature at 1.0 degrees C/min in a programmable methanol freezer. Embryos were seeded at -7 degrees C and then cooled to -25 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. After thawing and a one-step dilution of glycerol, embryos were cultured for 48 hr and viability was determined by blastocoel formation. Highest viability (70.0%) after 48 hr in culture was obtained for embryos frozen in 1.5 M glycerol plus 0.10 M trehalose as compared to 31% viability for embryos frozen with glycerol alone. These observations suggest that trehalose can be used in combination with glycerol as a cryoprotectant and that a high rate of viability can be achieved after a one-step dilution of the cryoprotectants.     

Successful direct transfer of vitrified sheep embryos

The use of a simple cryopreservation method, adapted to direct transfer of thawed embryos may help to reduce the costs of embryo transfer in sheep and increase the use of this technique genetic improvement of this species. Two experiments were made to test a vitrification method that is easy to apply in field conditions. All embryos were collected at Day 7 of the estrous cycle of FSH-stimulated donor ewes and were assessed morphologically, washed in modified PBS and incubated for 5 min in 10% glycerol, for 5 min in 10% glycerol and 20% ethylene glycol and were transferred into the vitrification solution (25% glycerol and 25% ethylene glycol). All solutions were based on mPBS. Embryos were loaded in straws (1 cm central part, the remaining parts being filled with 0.8 M galactose in mPBS) and plunged into liquid N2 within 30 sec of contact with the vitrification solution. The straws were thawed (10 sec at 20 degrees C) and the embryos were either transferred directly or after 5 min of incubation in the content of the straw (followed by washing in PBS) into the uterus of a recipient ewe. In Trial 1, the pregnancy rates at term (72 vs. 72%) as well as the embryo survival rates (60 vs 50% respectively) were not different between fresh (n = 48 embryos) and vitrified (n = 50) embryos. In a second trial no difference was observed between vitrified embryos transferred after in vitro removal of the cryoprotectant (n = 86 embryos) or directly after thawing (n = 72) both in terms of lambing rate (67 vs. 75%, respectively) and embryo survival rate (lambs born/embryos transferred; 49 vs. 53%). This method of sheep embryo cryopreservation provided high pregnancy and embryo survival, even after direct transfer of the embryos.     

Evaluation of cryopreservation techniques for bovine embryos

A total of 228 embryos was nonsurgically collected from superovulated cows and dehydrated in dimethyl sulfoxide (DMSO) or glycerol by a three-step procedure or a (T.I.T.) timed interval titration procedure. Embryos were loaded in straws, frozen by cooling to -6.0 degrees C at 1.0 degrees C/min, and seeded, followed by cooling to -30 degrees C at 0.3 degrees C/min and to -38 degrees C at 0.1 degrees C/min. At this time the straws were plunged into liquid nitrogen at -195 degrees C. Embryos were thawed in a 27 degrees C or 37 degrees C water bath and rehydrated by a six-step, three-step (sucrose) or one-step (sucrose) procedure. This yielded a 2 x 2 x 2 x 3 factorial treatment structure. Survival was based on development after 12 h in in vitro culture. The only significant single factor affecting survival was the initial quality grade of the embryo. Grades 1 and 2 embryos survived more often than Grade 3 embryos (P < 0.05). Using DMSO as the cryoprotectant resulted in better scores for the post dehydration to post thawing interval (P = 0.02). For both intervals, post dehydration to post thawing and post thawing to post rehydration, the previous quality grade was significant in determining the subsequent quality grade (P < 0.01). At each step of the freeze-thaw process, the embryos became progressively less morphologically intact.     

Survival of frozen mouse embryos after rapid thawing from -196 degrees C.

The effect of the rate of rewarming on the survival of 8-cell mouse embryos and blastocysts was examined. The samples were slowly cooled (0.3--0.6 degrees C/min) in 1.5 M-DMSO to temperatures between -10 and -80 degrees C before direct transfer to liquid nitrogen (-196 degrees C). Embryos survived rapid thawing (275--500 degrees C/min) only when slow cooling was terminated at relatively high subzero temperatures (-10 to -50 degrees C). The highest levels of survival in vitro of rapidly thawed 8-cell embryos were obtained after transfer to -196 degrees C from -35 and -40 degrees C (72 to 88%) and of rapidly thawed blastocysts after transfer from -25 to -50 degrees C (69 to 74%). By contrast, for embryos to survive slow thawing (8 to 20 degrees C/min) slow cooling to lower subzero temperatures (-60 degrees C and below) was required before transfer to -196 degrees C. The results indicate that embryos transferred to -196 degrees C from high subzero temperatures contain sufficient intracellular ice to damage them during slow warming but to permit survival after rapid warming. Survival of embryos after rapid dilution of DMSO at room temperature was similar to that after slow (stepwise) dilution at 0 degrees C. There was no difference between the viability of rapidly and slowly thawed embryos after transfer to pseudopregnant foster mothers. It is concluded that the behaviour of mammalian embryos subjected to the stresses of freezing and thawing is similar to that of other mammalian cells. A simpler and quicker method for the preservation of mouse embryos is described.     

Effects of varying the holding temperature and interval from collection to freezing on post-thaw development of bovine embryos in vitro

The objective of this study was to evaluate the in vitro development of frozen-thawed bovine embryos held at room temperature or refrigerated for 2, 6 or 12 h prior to freezing. After recovery, embryos were randomly assigned to be placed in holding media for 2 h (n=131), 6 h (n=136) or 12h (n=133) prior to freezing. Approximately one-half of the embryos were refrigerated (5 degrees C; n=203) while the remaining half were held at room temperature (22 degrees C; n = 197) until freezing. Embryos were frozen in 10% ethylene glycol and stored in liquid nitrogen. After thawing, embryos were cultured for 72 h in Ham's F-10 media supplemented with 4% fetal bovine serum. Embryos were evaluated for quality and stage of development prior to freezing and after culture. At the end of culture, it was determined if each embryo had developed beyond the stage recorded prior to freezing and if the embryo had hatched from the zona pellucida. The percentage of embryos that developed during culture was greater (P < 0.001) for Grade 1 (81%) than for either Grade 2 (65%) or Grade 3 (48%) embryos. Likewise, a greater proportion (P < 0.001) of Grade 1 embryos developed to hatched blastocysts (60%) than either Grade 2 (40%) or Grade 3 (24%) embryos. The holding temperature from collection to freezing did not influence embryo development, regardless of the interval from embryo collection to freezing. The proportion of embryos that developed to expanded blastocysts and hatched was greater (P < 0.005) for embryos held 2 h prior to freezing (64%) than for embryos held for 12 h (33%). Hatching rate of embryos held 6 h prior to freezing (54%) tended (P < 0.08) to be lower than the hatching percentage for embryos held for 2 h. Thus, post-thaw embryonic development was impaired the longer embryos were held prior to freezing and temperature during the interval from collection to freezing did not affect post-thaw development.     

Effect of endothelial cell growth supplement and fibroblastic growth factor on early mouse embryo development in vitro

This study was conducted to examine the effect of endothelial cell growth supplement (ECGS) and fibroblastic growth factor (FGF) on early mammalian embryo development in vitro. Two hundred mouse blastocysts were placed randomly in culture wells containing one of five treatments: 1) Ham's F-10, 2) Ham's F-10 + 10 mug ECGS, 3) Ham's F-10 + 10 ng ECGS, 4) Ham's F-10 + 100 ng FGF and 5) Ham's F-10 + 10 ng FGF. In all cases, media were supplemented with 10% (v/v) normal steer serum. Embryos were cultured at 37 C with an atmosphere of 5% O(2), 5% CO(2) and 90% N and development was recorded at 12 h intervals for the duration of culture. The percentage of embryos that developed to expanded blastocyst (94.8), hatching blastocyst (74.4) and hatched blastocyst (71.8) in Ham's F-10 media were not different from embryos cultured in all other treatments except Ham's F-10 + 10 mug ECGS. A decrease in the percentage of embryos reaching expanded blastocyst (44.7), hatching blastocyst (23.7) and hatched blastocyst (18.4) was observed in Ham's F-10 + 10 mug ECGS. Also, a significant (P<0.01) decrease in development scores at 24, 48 and 72 h was observed for embryos cultured in Ham's F-10 + 10 mug ECGS. No difference was observed in the mean time to reach different developmental stages among treatments. The data suggest that ECGS and FGF at the doses tested have no beneficial effect on early mouse embryo development in vitro and 10 mug of ECGS has inhibitory effect.     

Nuclear transfer of sand cat cells into enucleated domestic cat oocytes is affected by cryopreservation of donor cells

In the present study, we used the sand cat (Felis margarita) as a somatic cell donor to evaluate whether cryopreservation of donor cells alters viability and epigenetic events in donor cells and affects in vitro and in vivo developmental competence of derived embryos. In Experiment 1, flow cytometry analysis revealed that the percentage of necrosis and apoptosis in cells analyzed immediately after freezing/thawing (61 vs. 8.1%, respectively) was higher than that observed in frozen/thawed cells cultured for 18 h (6.9 vs. 3.3%, respectively) or 5 days (38 vs. 2.6%; respectively). The relative acetylation level of H3K9 was lower in frozen/thawed cells (5.4%) compared to that found in cultured cells (60.1%). In Experiment 2, embryos reconstructed with frozen/thawed cells had a lower cleavage rate (85%; day 2) than did embryos reconstructed with cultured cells (95%), while development to the blastocyst stage (day 8) was not affected by cell treatment (17.0% with frozen/thawed cells vs. 16.5% with cultured cells). In Experiment 3, pregnancy rates were similar between both cell treatments (32% with frozen/thawed cells vs. 30% with cultured cells), but the number of embryos that were implanted, and the number of fetuses that developed to term was lower for embryos reconstructed with frozen/thawed cells (1.2 and 0.3%, respectively) than those reconstructed with cultured cells (2.6 and 1.8%, respectively), while the number of fetuses reabsorbed by day 30 was higher (75%) for embryos reconstructed with frozen/thawed cells than those reconstructed with cultured cells (31%). A total of 11 kittens from cultured cells and three kittens from frozen/thawed cells were born between days 60 to 64 of gestation. Most kittens died within a few days after birth, although one kitten did survive for 2 months. In Experiment 4, POU5F1 mRNA expression was detected in 25% of blastocysts derived from frozen/thawed cells, whereas 88 and 87% of blastocysts derived from cultured cells and by in vitro fertilization, respectively, expressed POU5F1. We have shown that cell cryopreservation increased the incidence of necrosis and apoptosis and altered epigenetic events in donor cells. Consequently, the number of embryos that cleaved, implanted, and developed to term-gestation and POU5F1 expression in derived blastocysts indirectly was affected.     

Pregnancy rate and survival in culture of in vitro fertilized bovine embryos frozen in various cryoprotectants and thawed using a one-step system

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours (38.5 degrees C, 5% CO(2)) in modified TCM-199 medium supplemented with 5% superovulated cow serum (SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3 M methyl cellosolve (MC), 1.1 M diethylene glycol (DEG), 1.8 M ethylene glycol (EG), 1.6 M propylene glycol (PG) and 1.1 M 1, 3-butylene glycol (BG) solutions. They were then loaded into 0.25-ml straws, placed into an alcohol bath freezer at 0 degrees C, cooled from 0 degrees C to -6 degrees C at -1 degrees C/minute, seeded, held for 10 minutes, and cooled again at -0.3 degrees C or -0.5 degrees C/minute to -30 degrees C. Straws were then plunged and stored in liquid nitrogen. After thawing in 30 degrees C water, the embryos were rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred nonsurgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows: EG (50.0%), MC (53.6%), DEG (56.9%), PG (58.0%) and BG (11.5%). The survival rate of embryos cooled at -0.3 degrees C vs -0.5 degrees C/minute was not significantly different (P>0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of -0.3 degrees C/minute (64.6%, 31 48 ) than at -0.5 degrees C/minute (22.6%, 12 53 ). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows: MC (48%, 10 21 ); DEG (30%, 3 10 ); EG (74%, 20 27 ); and PG (40%, 4 10 ). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF bovine embryos.     

Preliminary studies on bovine embryo survival following short-term storage at 4 degrees C

A total of 113 non-surgically collected bovine embryos, 5-8 days of age, were stored for 48 hours at 4 degrees C in a modified phosphate-buffered saline solution (PBS). Following storage, embryos were cultured for 8-12 hours at 37 degrees C, and those which were morphologically normal were transferred to synchronized recipients by several methods designed to achieve twin pregnancies. Embryos which were collected and transferred on the same day served as controls. Of 113 embryos stored, 47 (42%) appeared to be transferable after the brief culture period. There was a marked breed effect on viability after refrigeration, with Hereford embryos surviving significantly better than Angus embryos (71% vs. 12%, respectively, p < .001). Post-transfer embryo survival of stored and control embryos, based on actual calvings, was 34 and 48 percent, respectively, a difference which was not significant (p=0.3). A marked difference in pregnancy rate following non-surgical transfer by 2 different technicians was noted (50% vs. 21.7%, respectively).     

Cryopreservation of mouse embryos at -196 degrees C by vitrification

Embryos (8-16 cell) were obtained from random bred albino mice (6-8 weeks old) that were induced to superovulate by injections of 5 I.U. PMSG and 5 I.U. hCG given 48 hr apart. Embryos were exposed to intracellular cryoprotecting medium (glycerol 10%, 1-2 propanediol 20% in PBS) for 10 min and then transferred to extracellular vitrification medium (25% glycerol, 25% 1-2 propanediol in PBS). Vitrification medium containing embryos, and diluent (1 M sucrose) were loaded in a straw and immediately plunged into liquid N2. After thawing at 20 degrees C, the contents of the straw were mixed by shaking (1 step dilution) and emptied in a petri dish. After 3 washings in culture medium the embryos were kept in CO2 incubator for further development. In 3-step dilution procedure the dilution of cryoprotectants was done in 0.5 and 0.25 M sucrose before culture. Embryos in 3-step dilution of cryoprotectants exhibited high survival as compared to 1-step dilution (20.23% vs 6.55%).     

Effect of reduced concentrations of glycerol and various macromolecules on the cryopreservation of mouse and cattle embryos

The effect of different macromolecules [bovine serum albumin (BSA), Pluronic F-68, (ET surfactant), or sodium hyaluronate (SH)] on postthaw survival of mouse morulae and in vivo- and in vitro-derived bovine blastocysts frozen in 10, 5, or 1% glycerol solutions was investigated. Embryos were equilibrated with cryoprotectant solution at 25 degrees C for 10 min, seeded at -5 degrees C, cooled at 0.5 degrees C/min to -35 degrees C, and plunged into liquid nitrogen. Embryos were thawed in a 35 degrees C water bath, glycerol was removed with 0.6 M sucrose at 25 degrees C for 5 min, and postthaw viability was evaluated after 1, 24, and 48 h in culture. The addition of BSA supplementation improved postthaw survival of mouse morulae frozen in 5% glycerol, but not in 10% glycerol. All three macromolecular supplements were effective in increasing survival of mouse morulae in 5% glycerol but only BSA and SH were effective in increasing postthaw survival of in vivo- and in vitro-derived bovine blastocysts. None of the macromolecular supplements improved postthaw survival of embryos frozen in 1% glycerol.     

Culture of 5-day horse embryos in microdroplets for 10 to 20 days

Embryos were recovered from the uteri of mares 5 d after ovulation. Six embryos, all morulae, were placed singly in 200-ul droplets of Ham's F-12 with 10% fetal calf serum and cultured at 37 degrees C in a 5% CO(2) atmosphere. The embryos expanded to form blastocysts by the third day of culture. The blastocysts hatched from their zona pellucida, rather than the zona thinning and flaking off, as occurs in vivo. Hatching from the zona pellucida began on the third day of culture and was complete in five of six embryos by the sixth day. The embryonic capsule, normally present in equine embryos after Day 6, was not seen in the cultured embryos. The blastocysts continued to expand until 15 to 17 d of age (10 to 12 d in culture), reaching an average diameter (+/- SD) of 2052 +/- 290 um, after which time they either collapsed or contracted. These results demonstrate that equine embryos can be maintained in long-term culture in vitro, exhibiting continued growth and expansion in the absence of the embryonic capsule.