Effect of removing the zona pellucida on development of hamster and bovine embryos invitro and invivo

Uterine stage embryos collected from the hamster (8-cell) and cow (morula, early blastocyst) were monitored for development $\text{in}$$\text{vitro}$ (embryo culture) and $\text{in}$$\text{vivo}$ (embryo transfer) following premature removal of the zona pellucida.Removal of the zona pellucida did not significantly affect $\text{in}$$\text{vitro}$ development to the blastocyst stage of (1) 8-cell hamster embryos (zonae removed by a combined enzymic-mechanical procedure), (2) bovine morulae (zonae removed by mechanical means only) (3) early bovine blastocysts (zonae removed by the enzymic-mechanical technique).Zona-free hamster embryos formed significantly fewer viable fetuses than did zona-intact embryos. The lower incidence of fetal development observed following transfer of zona-free 8-cell hamster embryos may have resulted in part from the formation of chimeras by fusion of these embryos $\text{in}$$\text{utero}$. Such fusion was observed to occur $\text{in}$$\text{vitro}$ between zona-free embryos placed in close proximity. The proportion of pregnancies resulting from transfer of bovine blastocysts cultured from zona-free morulae was similar to that of zona-intact embryos.In this study we have demonstrated that (1) enzymic and mechanical procedures used to remove zonae pellucidae from uterine-stage hamster and bovine embryos do not adversely affect subsequent development of these embryos $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ and (2) zonae pellucidae are not required for normal development of these embryos. These findings have implications for microsurgery of mammalian embryos and for embryo transfer.     

Effect of lecithin on in vitro and in vivo survival of in vitro produced bovine blastocysts after cryopreservation

The use of soybean lecithin in an glycerol-based solution for slow freezing of in vitro matured, fertilized and cultured (IVMFC) bovine embryos was examined. Embryos were developed in vitro in INRA Menezo's B2 medium supplemented with 10% fetal calf serum (FCS) on Vero cells monolayers. Day 7 blastocysts were frozen in a two-step protocol consisting of exposure to 5% glycerol and 9% glycerol containing 0.2 M sucrose in F1 medium + 20% FCS. Soybean lecithin was either added or not to the freezing solutions at a final concentration of 0.1% (w/v). In Experiment 1, blastocysts were equilibrated in cryoprotectant solutions without cooling. Cryoprotectant was diluted from embryos with 0.5 M and 0.2 M sucrose. The percentages of fully expanded and hatched blastocysts treated with or without lecithin after 24 and 48 h in culture were not significantly different (100 versus 100% and 93.3 versus 100%, respectively). In Experiment 2, the in vitro survival of frozen-thawed IVMFC blastocysts was compared when cryoprotectant solutions were either supplemented or not with lecithin. No significant effect of lecithin was found on the ability of frozen-thawed blastocysts to re-expand after 48 h in culture (65.6 and 54.2%, respectively). However, the post-thaw hatching rate of embryos cryopreserved in the presence of 0.1% lecithin was significantly higher after 72 h in culture (52 and 31.8%, respectively). In Experiment 3, the ability of frozen-thawed IVMFC blastocysts to establish pregnancy following single embryo transfer was determined. Transfers of 58 and 66 frozen-thawed embryos cryopreserved with or without lecithin resulted in 6 and 10 (10.3 and 15.1%, respectively) confirmed pregnancies at Day 60. Addition of lecithin to cryoprotectants did not improve the in vivo development rate of cryopreserved IVMFC bovine blastocysts.     

Survival rates and sex ratio of bovine IVE embryos frozen at different developmental stages on day 7

Two experiments were designed to determine the effects of stage of development on Day 7 of in vitro-produced bovine embryos on survival after deep freezing and on sex ratio. Bovine IVF embryos and bovine oviductal epithelial cells (BOEC) were co-cultured in TCM-199 and, on Day 7 after insemination (Day 0), were morphologically evaluated and divided into groups by developmental stage. In Experiment 1, embryos classified as early blastocysts, blastocysts and full-expanding blastocysts were randomly subdivided into 2 groups by replicate: 50% of the embryos were placed immediately in a new BOEC co-culture (fresh group), while the other 50% were frozen, thawed and placed in a new BOEC co-culture (frozen/thawed group). Embryos were frozen in 1.5 M glycerol using a standard slow cooling technique. Fresh and frozen/thawed embryos were compared for survival rate (embryos hatching/hatched) in BOEC co-culture over the following 3 d (i.e., Days 7 to 10). The overall survival of the 425 embryos (early to full-expanding blastocysts) was 33% and was not different between fresh (35%) and frozen/thawed (30%) embryos. Survival of embryos cultured fresh or after freezing/thawing was higher for full-expanding blastocysts than for early blastocysts or for blastocysts, both of which were not different. In Experiment 2, all frozen/thawed embryos used in Experiment 1 plus all morulae and hatched blastocysts collected and frozen on Day 7 without regard to survival were sexed utilizing the polymerase chain reaction (PCR) technique. Sex of the embryos, by stage of development on Day 7, was determined in order to compare the rate of development in BOEC co-culture with the sex ratio (percentage of males). A total of 235 embryos was sex-determined with an overall percentage of males of 51%, which was not different from the expected 1:1 sex ratio. Both full-expanding blastocysts and hatched blastocysts had a significantly higher (P < 0.05) proportion of males (68 and 100%, respectively), while morulae had a significantly lower proportion of males (24%). Early blastocysts and blastocysts did not differ from a 1:1 sex ratio. The results indicate that male embryos develop faster in vitro than female embryos. The higher survival rate of full-expanding blastocysts after freezing/thawing, and the production of a higher number of males than females among embryos of this developmental stage suggest that a greater number of male fetuses may result from the successful freezing and transfer of in vitro-produced bovine embryos.     

Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol

Survival of IVF-derived bovine embryos of different ages and stages of development, produced in 2 different co-culture systems and frozen in 2 different cryoprotectants, was investigated. In vitro-derived bovine embryos (n = 5,525) were utilized to study survival following exposure to cryoprotectants and after freezing. Survival of the frozen embryos was based on blastocyst re-expansion 24 h and hatching 72 h after thawing. There was no difference in survival when embryos were exposed to either glycerol (Gly) or ethylene glycol (EG) for 10 or 40 min with the cryoprotectant diluted with or without freezing. In 2 of 3 experiments in which a comparison was possible, more blastocysts frozen in 1.4 M glycerol than in 1.5 M ethylene glycol survived. Addition of 0.25 M sucrose to 1.5 M ethylene glycol in the freezing solution did not improve embryo survival. More blastocysts frozen on Day 7 of in vitro culture survived than those frozen on Day 6 or Day 8. On Days 6, 7 and 8, embryos in the most advanced stage of development survived better than those at less advanced stages. Post-thaw survival did not differ for embryos produced in co-culture with Buffalo Rat Liver (BRL) cells with either Menezo B2 Medium or Tissue Culture Medium 199 and frozen in 1.4 M glycerol.     

Direct freezing of cattle embryos after partial dehydration at room temperature

A new and simple method for freezing of bovine morulae and blastocysts was developed. Embryos were predehydrated at room temperature, frozen at -30 degrees C (cooling rate = 12 degrees C/min), and plunged into liquid nitrogen. This method was compared in vitro and in vivo to the slow freezing method (0.3 degrees C/min to -30 degrees C). Predehydration of the embryos in 1.5M glycerol was achieved by sucrose solution that makes the cells osmotically shrink. After the predehydrated morulae and blastocysts were frozen and thawed, 6 .4% (33 52 ) were developed in vitro for 48h and 44.2% (23 52 ) were hatched. Development obtained with slowly frozen embryos were 70.8% (17 24 ) and 58.3% (14 24 ) respectively. After transfer to recipient heifers, 33.3% (7 21 ) of the embryos frozen according this new method developed normally into viable foetuses or calves. This was the case for 48.5% (16 33 ) of the slowly frozen embryos.     

Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro

This study compares the effects of conventional controlled-rate freezing and vitrification on the morphology and metabolism of in vitro-produced bovine blastocysts. Day 7 expanded blastocysts cultured in synthetic oviduct fluid with 5% fetal calf serum were frozen in 1.36 M glycerol, 0.25 M sucrose or vitrified in 25% glycerol, 25% ethylene glycol. Cell alterations and in vitro development were evaluated immediately after thawing or after 72 h. The effect of cryopreservation on inner cell mass and trophectoderm (TE) cell number as well as glucose, pyruvate, and oxygen uptakes, and lactate release by blastocysts were evaluated. Immediately after thawing, blastocysts showed equivalent cell membrane permeabilization after both cryopreservation procedures, while alterations in nuclear staining were more frequent in vitrified embryos. After culture, similar survival and hatching rates were observed. Both procedures decreased cell number immediately after thawing and after 72 h. However, the number of TE cells was lower in frozen embryos than in vitrified ones. In relation to this, frozen blastocysts showed a decrease in glucose, pyruvate, and oxygen uptake, although those parameters were not altered in vitrified embryos. An increased glycolytic activity was also observed in frozen embryos, indicating a stress response to this procedure.     

Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability

To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Vero cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 +/-45.6 nuclei compared to 137.5+/-32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freezing/thawing, delipidated (n=73), control (n=67) and sham (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.     

Effect of dimethyl sulfoxide and protein concentration on the viability of two-cell mouse embryos frozen with a rapid freezing technique

Two-cell mouse embryos were frozen by direct plunging into liquid nitrogen after a 3-min exposure to solutions containing 0.25 M sucrose with 1.5, 3, or 4.5 M dimethyl sulfoxide (Me2SO), and 0, 4, 8, 16, or 32 mg/ml bovine serum albumin (BSA). In the absence of BSA, significantly more embryos were lost or damaged during freezing and thawing. Increasing the BSA concentration from 4 to 32 mg/ml had no significant effect on subsequent embryo viability in vivo or in vitro. Blastocyst formation in vitro was greater than 90% in embryos exposed to the cryoprotective solutions only. Although development to blastocysts was not significantly different to nonfrozen controls in most groups frozen in 3 and 4.5 M Me2SO (up to 92% blastocysts), it was significantly reduced when embryos were frozen in 1.5 M Me2SO (up to 65% blastocysts). The development to fetuses of embryos frozen in 3 M Me2SO (64 to 74% fetuses) was not significantly different from nonfrozen controls (68 to 79% fetuses) or embryos frozen by a conventional slow cooling method (70%). Frozen thawed two-cell embryos developed into normal adults which were able to reproduce normally. We conclude that this freezing method can efficiently cryopreserve early cleavage stage mouse embryos.     

Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification

The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0.05) were obtained by vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.     

Blastocyst viability and generation of transgenic cattle following freezing of in vitro produced, DNA-injected embryos

This study examined whether the viability, determined in vitro, of DNA-injected bovine embryos produced in vitro was affected by freezing, and if the frozen embryos developed to term following transfer to recipients. In vitro fertilized zygotes were injected with the pBL1 gene and then co-cultured with mouse embryonic fibroblasts (MEF) in CR1aa medium. Embryos were prepared for cryopreservation by exposure to a 10% (v/v) glycerol solution, loaded into 0.25 ml straws and then frozen by conventional slow freezing. Thawing was by rapid warming in water (37 degrees C) and embryos were rehydrated in PBS diluents of 6%, 3% and 0% (v/v) glycerol supplemented with 0.25 M sucrose and 0.5% (w/v) BSA. In Experiment 1, blastocysts that developed from DNA-injected embryos were individually classified into three morphological groups and three stages of development prior to freezing. DNA-injected blastocysts of excellent quality at freezing showed a higher survival rate (78.8+/-10.6%) after thawing than those of good (60. 9+/-16.4%) or fair (12.5+/-5.9%) quality (P<0.05). Post-thaw survival rate, judged in vitro, increased with more advanced stage of blastocyst development at freezing (early 48.8+/-15.9%, mid 52. 1+/-12.6% and expanded 71.2+/-1.1; P<0.05). In Experiment 2, the frozen/thawed embryos were transferred to recipients to examine in vivo viability. Following transfer of one or two embryos per recipient, pregnancy rates at 60 days of gestation were 13.6% (13/96) for frozen embryos and 26.5% (43/162) for fresh embryos (P<0. 05). Of the 12 live calves born from the frozen/thawed embryos, two males (18.3%) were transgenic. None of the live-born calves derived from fresh embryos exhibited the transgene. One of transgenic bulls did not produce transgenic sperm. Three out of 23 calves (13.0%) produced from cows inseminated with semen of the other bull were transgenic, suggesting that this animal was a germ-line mosaic. These studies indicated that the viability of in vitro produced, DNA-injected bovine blastocysts was affected by freezing and by both the quality and stage of development of the embryo prior to freezing. The generation of transgenic cattle demonstrates that it is feasible to freeze DNA-injected, in vitro produced embryos.     

Freezability of porcine blastocysts at different peri-hatching stages

The freezability of porcine peri-hatching stage blastocysts was investigated by the cryopreservation of embryos at -196 degrees C with 1.5 M glycerol and by thawing, followed by in vitro culture. Of 66 expanded blastocysts frozen, 34 (51.5%) developed in vitro after thawing, while only 2 (6.7%, P<0.05) of 30 earlier stage blastocysts survived freezing. After freezing of 85 hatched blastocysts with an embryonic diameter of 150 to 300 mum, 59 (69.4%) surviving embryos were obtained, whereas none of the 78 advanced staged hatched blastocysts (>300 mum) survived the cryopreservation. High post-thaw survival (32 39 , 82..1%) was obtained with in vitro-hatched blastocysts precultured in Whittingham's M-16 medium containing 12mg/ml bovine serum albumin (BSA). In contrast, none of the 14 in vitro-hatched blastocysts precultured in the M-16 medium supplemented with 15% fetal calf serum (FCS) survived freezing. Similarly 51 of 56 hatced blastocysts (diameter = 150 to 300 mum) precultured in the M-16 medium supplemented with BSA survived cryopreservation, compared with 3 of 26 embryos precultured in the medium supplemented with FCS (P<0.001). Because both groups of the embryos precultured with BSA or FCS possessed normal ability to develop after transfer (developmental rate = 61.1 and 93.3%), the improved freezability of the embryos precultured with BSA may relate to a favorable change of embryonic cell membranes during the culture period. It was concluded that in vitro-hatched blastocysts precultured in medium containing BSA and in vivo-hatched blastocysts at the appropriate stage of development could both tolerate deep freezing to -196 degrees C; however, a differece in the freezability of embryos between breeds of pig was suggested from a further experiment performed with German Landrace embryos.     

Survival of early preimplantation porcine embryos after co-culture with cells producing an avian retrovirus

To determine the relative survival of porcine embryos after co-culture with cells producing an avian retrovirus, four-cell stage embryos were obtained from sows following synchronization with altrenogest and superovulation with gonadotropins. These embryos were randomly assigned to the following treatments: no manipulation (zona-intact); zona removed with acidified Tyrode's solution (zona-free); and zona removed followed by co-culture with D-17 canine cells producing an avian retrovirus vector derived from spleen necrosis virus (zona-free + co-culture). The survival rates of four-cell stage embryos to morulae or early blastocysts during a 48-h culture period were 93.3, 80.0 and 57.7% in zona-intact, zona-free and zona-free + co-culture groups, respectively. Following embryo transfer, the development of embryos to fetuses at six weeks of gestation was 37.5, 30.0 and 11.7% in zona-intact, zona-free and zona-free + co-culture groups. These results indicate that early preimplantation porcine embryos can develop to apparently normal fetuses following co-culture with cells producing a retrovirus, and the feasibility of this method for retrovirus-mediated gene transfer in pigs was demonstrated.     

Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing

Factors affecting viability of IVF-derived bovine blastocysts after freezing and thawing were investigated. A total of 1,101 ova matured and fertilized in vitro were cultured under 2 different conditions, 1) in TCM-199 on granulosa cell monolayers at 5% CO(2) in air and 2) in synthetic oviduct fluid (SOF) medium without somatic cell support at 5% CO(2), 5% O(2), 90% N(2). All blastocysts that developed from the 2 different culture systems were individually classified into 4 grades of embryo quality and were then frozen by conventional slow freezing. Developmental rates of the IVF-derived ova to blastocysts and the survival rates of the frozen-thawed blastocysts were not different between the SOF medium (16 and 49%) and the co-culture system (13 and 61%, respectively). Survival of frozen-thawed blastocysts was affected by embryo quality in both the SOF and co-culture systems (P<0.001). Blastocysts produced in vitro were also individually classified into 3 developmental stages and were then cultured for 3 d in the co-culture system with granulosa cells after freezing and thawing. There was a difference in the survival rate of frozen-thawed embryos between blastocyst developmental stages (early vs mid, P<0.05; mid vs expanded, P<0.01; early vs expanded, P<0.001). The post-thawing survival rate of blastocysts frozen at Day 7 (62%) of culture was higher compared with that of Day 8 (45%), but there was no difference in survival rate between Day 7 and 8 of culture. The results indicate that the quality and developmental stage of blastocysts are important factors influencing their survival after freezing and thawing.     

Removal of swine zonae pellucidae with sequential exposures to pronase and acidified medium

Exposure to acidified PBS (pH 3) for 60 sec removed swine zonae pellucidae from 70.4% of 27 swine morulae, and 73.7% of these formed blastocysts in culture. Further investigations revealed that treating embryos with 0.5% pronase and acidified PBS (pH 3) for 30 sec each was more effective. Zonae were removed in 90.6% of 85 embryos (four-cell to morulae) treated. A total of 76.9% of 65 zona-free embryos and 81.6% of 38 untreated embryos formed blastocysts (P > 0.05). An additional 57 untreated and 49 zona-free embryos (morulae to blastocysts) were transferred to seven recipient sows. Four sows returned to estrus (18 to 27 days), but three others were pregnant when slaughtered at 38 to 42 days. One pregnant sow had received a combination of five zona-free and six untreated embryos and demonstrated the potential for further development of treated embryos in vivo.     

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.     

Effect of human leukemia inhibitory factor on in vitro development of IVF-derived bovine morulae and blastocysts

This study was conducted to investigate whether human leukemia inhibitory factor (hLIF) improves the subsequent development of IVF-derived bovine morulae and blastocysts. To obtain IVF-derived bovine morulae, ova were matured and fertilized in vitro and cultured in 0.5 ml of synthetic oviduct fluid (SOF) medium supplemented with 10% human serum (HS) for 5 d at 39 degrees C under a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2). Morulae and early blastocysts at Day 5 of culture were cultured in 0.5 ml of SOF medium with or without 5000 U/ml recombinant hLIF for 2 or 3 d (2 groups). To investigate the effect of addition of hLIF on the subsequent development of morulae, SOF medium was supplemented with 8 mg/ml BSA instead of HS. To test whether hLIF affects the subsequent development of IVF-derived bovine blastocysts, only good blastocysts that developed from SOF medium with or without hLIF at Days 7 and 8 of culture were frozen by a conventinal slow freezing method and again cultured in SOF medium with or without the addition of hLIF for 3 d after thawing (4 groups). Survival of frozen-thawed bovine embryos was evaluated for re-expansion and hatching of blastocysts during 3 d of culture. There was no significant difference in the developmental rate of Day 5 embryos to blastocysts between those cultured with (47.8%) and without (47.6%) addition of hLIF. However, the addition of hLIF before freezing significantly increased the hatching rate of IVF-derived bovine morulae (P < 0.05), whereas addition of hLIF after thawing did not increase the subsequent development of blastocysts. These results suggest that hLIF added at the Day 5 morula stage may contribute to bovine embryonic development through the hatching process.     

Survival of rapidly frozen hatched mouse blastocysts

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [approximately 1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum +0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.     

Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification

In this study, the cryoprotectant ethylene glycol (EG) was tested for its ability to improve and facilitate the cryopreservation of in vitro produced (IVP) bovine embryos. Embryos were cryopreserved in EG solutions supplemented with either newborn calf serum (NBCS) or polyvinyl alcohol (PVA). To assess EG toxicity, the embryos were equilibrated in EG concentrations from 1.8 to 8.9 M at room temperature for 10 min and then cultured for 72 h on a cumulus cell monolayer. The hatching rate was highest for day 7 blastocysts frozen in 3.6 M EG (98%) and was not different from the control group (85%). The controlled freezing (0.3 degrees C/min to -35 degrees C) of expanded day 7 blastocysts resulted in a hatching rate of 81%, which was similar to that of the nonfrozen controls (76%). Differential staining revealed only very few degenerate blastomeres attributed to freezing and thawing. Upon direct nonsurgical transfer of day 7 expanded blastocysts frozen in 3.6 M EG, a pregnancy rate of 43% was achieved, while the pregnancy rate after transfer of other developmental stages was significantly lower (22% with expanded day 8 blastocysts). When bovine IVP embryos were incubated at room temperature in 7.2 M EG preceded by preequilibration in 3.6 M EG, the hatching rate of day 7 expanded blastocysts reached 93%. Upon vitrification of IVP day 7 and day 8 blastocysts and expanded blastocysts in 7.2 M EG, the latter showed a higher hatching rate (42%) than blastocysts (12%). Overall, PVA as supplement to the basic freezing solution instead of NBCS had deleterious effects on survival after controlled freezing or vitrification. The simple cryopreservation protocol employed in this study and the low toxicity of ethylene glycol highlight the usefulness of this approach for controlled freezing of IVP embryos. However, further experiments are needed to improve the pregnancy rate following embryo transfer and to enhance survival after vitrification.     

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows?. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.     

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.