Efficient cryopreservation of bovine blastocysts derived from nuclear transfer with somatic cells using partial dehydration and vitrification

Preservation by vitrification of Day 7 and Day 8 bovine blastocysts derived from nuclear transfer with cumulus cells was compared with preservation of in vitro fertilized blastocysts. In Experiment 1, embryos were vitrified in PBS containing 60% ethylene glycol. In Experiment 2, they were vitrified in combination with partial dehydration using a solution of 39% ethylene glycol + 0.7 M sucrose and 8.6% Ficoll. In Experiment 1, survival and hatching rates were 44 and 95% for nuclear transferred embryos, and 78 and 55% for in vitro fertilized embryos, respectively. In Experiment 2, survival and hatching rates were 93 and 95% for nuclear transfer embryos, and 77 and 85% for in vitro fertilized embryos, respectively. It is concluded that Day 7 and Day 8 bovine blastocysts derived from cumulus cells could be cryopreserved without the loss of viability by a simple and efficient method using a combination of partial dehydration and vitrification.     

Osmotic behavior of in vitro produced bovine blastocysts in cryoprotectant solutions as a potential predictive test of survival

The osmotic behavior of bovine blastocysts produced in vitro was filmed during exposure to and dilution of cryoprotectant solutions used for vitrification. The relationship between the changes in the diameter of embryos and their subsequent survival was assessed. Embryos collected on Day 6 and Day 7 postinsemination were exposed to 10% glycerol (GLY) for 5 min, 10% GLY + 20% ethylene glycol (EG) for 5 min, and 25% Gly + 25% EG for 30 s, before dilution in 0.85 M galactose and finally in embryo transfer freezing medium (ETF). Embryos that had a higher probability of survival behaved as perfect osmometers, shrinking, reexpanding, or swelling according to an identical pattern, whereas embryos that deviated from this standard usually did not survive. The initial embryo diameter, duration of shrinkage and expansion in 10% glycerol, duration of reexpansion in ETF, and final embryo diameter were clearly predictive of the ability to hatch after culture in vitro. On a given day postinsemination, larger blastocysts were more likely than smaller blastocysts to survive and hatch after exposure to cryoprotectants with or without vitrification.     

Survival of vitrified sheep embryos in vitro and in vivo

The effects of the composition of vitrification media, the duration of exposure to the media and the stage of development were examined on the survival of vitrified Day-6 sheep embryos. Vitrification media that contained two cryoprotectants in equal molar concentrations were used. In Experiment 1, the effects of the types (glycerol + propylene glycol or glycerol + ethylene glycol) and concentrations (3.5 + 3.5 or 4.5 + 4.5 M) of cryoprotectants and the level of BSA supplementation (0.4 or 20%) were investigated in a 2 x 2 x 2 design. The embryos were exposed to vitrification media for 30 sec at 18 to 24 degrees C before vitrification. The in vitro survival rate was not affected by the level of BSA supplementation, but there was an interaction between the types and concentrations of cryoprotectants used (P<0.01). Embryos cryopreserved in mixtures of glycerol + propylene glycol survived better when the concentration of cryoprotectants was 3.5 M while the survival of embryos cryopreserved in mixtures of glycerol + ethylene glycol was higher at 4.5 M cryoprotectant concentration. In Experiments 2 and 3, the effect of the duration of exposure (15, 30, 60 or 120 sec) to vitrification media at 4 to 12 degrees C was investigated on the survival rate in vivo. Vitrification media contained 3.5 M glycerol + 3.5 M propylene glycol or 4.5 M glycerol + 4.5 M ethylene glycol in Experiments 2 and 3, respectively. The survival rate in vivo, increased when the duration of exposure to vitrification media was increased from 15 to 30 sec, but the viability declined when the duration of exposure was further increased to 60 (Experiment 3) or to 120 sec (Experiment 2). The effect of the stage of development was significant only in Experiment 1 (P = 0.032), but in all three experiments the rate of survival increased with advancing stages of development from late morulae to late blastocysts. The best result was achieved in Experiment 2, when embryos were exposed to a mixture of 3.5 M glycerol + 3.5 M propylene glycol for 30 or 60 sec. Under these conditions 52% (22 42 ) of rapidly cryopreserved sheep embryos developed into lambs. This result shows that a simple rapid procedure for the cryopreservation of sheep embryos can produce a survival rate comparable to that obtained using more complex traditional procedures.     

Effect of sugars-addition on the survival of vitrified bovine blastocysts produced in vitro

We investigated the effect of addition of sugars to a vitrification solution on the survival rate of bovine blastocysts produced in vitro. In vitro-matured (IVM) and in vitro-fertilized (IVF) bovine Day-6 to Day-8 bovine blastocysts were classified into 3 developmental stages: early blastocysts, blastocysts and expanded blastocysts. The blastocysts were cryopreserved in 1 of 3 vitrification solutions: 1) 25% glycerol25% ethylene glycol (GE); 2) 20% glycerol20% ethylene glycol3/4 M sucrose (GES); and 3) 20% glycerol20% ethylene glycol3/8 M sucrose3/8 M dextrose (GESD). The basic solution was Dulbecco's PBS supplemented with 20% of fetal calf serum. Embryos were exposed to each vitrification solution in 3 steps, and after loading into 0.25-ml straws, were plunged into liquid nitrogen. After warming in water bath at 20 degrees C, cryoprotectants were diluted in 1/2 M and 1/4 M sucrose each for 5 min. Equilibration and dilution procedure except warming were conducted at room temperature (23 to 27 degrees C). After dilution, the embryos were cultured in Ham's F10 medium0.1 mM beta-mercaptoethanol20% fetal calf serum. Survival rates of embryos at 48 h of incubation of each of the 3 developmental stages (early blastocysts, blastocysts and expanded blastocysts) exposed to the 3 types of the vitrification solutions (GE, GES and GESD) were 23.5, 33.3, 65.8% (early blastocysts, blastocysts and expanded blastocysts respectively) in GE, 55.6, 71.9, 90.5% in GES and 84.6, 83.3, 95.8% in GESD respectively. These results indicate that a mixture of 25% glycerol25% ethylene glycol is not suitable for vitrification of early bovine blastocysts; however, addition of sugars to the solution significantly (P<0.01) improved the survival rate of the vitrified blastocysts, independently of their stage of development.     

Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro

Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar.     

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.     

Successful cryopreservation of expanded equine blastocysts

Effective cryopreservation of expanded equine blastocysts (> 300 μm in diameter) has been difficult, perhaps due to the volume of blastocoele fluid or the presence of the equine embryonic capsule. Recently, we reported normal viability of equine embryos after trophoblast biopsy, which resulted in blastocyst collapse. The present study addressed the effect of biopsy and resultant breach of the capsule and blastocyst collapse on survival of expanded equine blastocysts after vitrification. First, non-biopsied, small embryos (< 300 μm) were vitrified in fine-diameter microloader pipette tips using dimethylsulfoxide-containing medium (DM) or ethylene glycol-containing medium (EG). A third group was vitrified with EG, but was warmed using sucrose (EG/s). Embryos in the DM and EG/s treatments grew in culture after vitrification, and established pregnancies after transfer (3 of 12 and 3 of 6, respectively). Expanded blastocysts 300-730 μm in diameter were then biopsied and vitrified; rates of normal pregnancy (detection of embryonic heartbeat) after warming and transfer were 2 of 16 (13%) and 6 of 13 (46%) for DM and EG/s treatments, respectively (P = 0.05). Within the EG/s treatment, it appeared that greater loss of blastocoele fluid after biopsy was associated with higher survival. Therefore, an altered (“Central”) biopsy technique was used to aspirate blastocoele fluid, followed by vitrification in EG/s. Pregnancy rates were 1 of 8 (13%) for embryos cultured after warming and 4 of 7 (57%) for embryos transferred immediately after warming (P = 0.1). Finally, expanded blastocysts 407 to 565 μm in diameter were biopsied from the periphery, and blastocoele fluid was removed with gentle suction. After vitrification with EG/s, this resulted in a rate of normal pregnancy of 5 of 7 (71%). These findings demonstrated that blastocoele collapse and vitrification in fine-diameter pipettes allowed successful cryopreservation of expanded equine blastocysts.     

Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts

A series of five experiments measured the high survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. The vitrification solution (designated VS) contained 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline. Embryos developed in vitro at Days 7 and 8 (Day 0 = insemination day) were exposed in one step to VS for 1 min or two steps with 10% ethylene glycol for 5 min and then VS for 1 min. In both cases, the embryos were finally cryopreserved in liquid nitrogen. After the embryos were warmed rapidly and the VS solution diluted, the survival rates were assessed by monitoring hatching rate in vitro. They were 13.0% for the one-step and 72.7% for the two-step procedures (P < 0.001). When embryos were exposed to individual solutions containing 6% (w/v) of each of 4 macromolecules (polyethylene glycol, BSA, polyvinylpyrrolidone or Ficoll) in the two-step protocol and then cryopreserved, the survival rates were 79.3, 34.8, 41.4 and 57.1%, respectively. After embryos had been exposed to the VS in two steps and then cryopreserved, there were no significant differences in survival rates when the solutions were diluted with or without sucrose. These results indicated that a vitrification solution containing polyethylene glycol can be used for cryopreservation of bovine blastocysts produced in vitro, and that a two-step addition of VS improved the in vitro survival of post-warming embryos. It was also shown to be possible to dilute post-warming embryos directly without the use of sucrose solution.     

Investigation of cryoprotectant toxicity to porcine embryos

The objective of this study was to examine the potential toxicity of sucrose (Experiment 1) and of various cryoprotectants (Experiment 2) to porcine preimplantation embryos. In Experiment 1, 65 embryos, ranging from compact morulae to hatched blastocysts, were allocated within donor female across 5 concentrations of sucrose (0, 0.25, 0.50, 1.0, 2.0 M) to determine the highest concentration that would not inhibit subsequent embryo development. After a 48-h post-treatment culture period, the embryos were stained and cell nuclei were counted. The concentration of sucrose affected embryo development (P < 0.001) and embryo quality (P < 0.001). Embryos placed into 2.0 M sucrose exhibited poorer development and quality than embryos at the lower 4 concentrations, which were not different from one another. In Experiment 2, 182 embryos of the same developmental stages as in Experiment 1 were collected from 16 donors. Embryos were allotted within donor female to 2 of the 5 concentrations (10, 20, 30, 40, or 50%) of each of 3 cryoprotectants (ethylene glycol, propylene glycol, glycerol). After a 30-sec exposure to a cryoprotectant, the embryos were cultured and stained as in Experiment 1. As the concentration of an individual cryoprotectant increased beyond 30%, embryo development decreased. Embryos exposed to glycerol or propylene glycol exhibited poorer development than did embryos placed into ethylene glycol, especially at concentrations of 40% or higher.     

Successful cryopreservation of mouse blastocysts using a new vitrification solution.

Mouse blastocysts were exposed to solutions containing four concentrations (10, 20, 30 and 40% v/v) of six permeating cryoprotectants (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide, 1,3-butanediol and 2,3-butanediol) in phosphate-buffered saline (PBS) with calf serum (CS) at room temperature (20-22 degrees C). Blastocysts were exposed to these solutions for various periods, diluted into PBS plus CS with or without 1 mol trehalose l-1 solution and their subsequent survival in vitro was examined. Two-way anova showed a significant interaction (P < 0.01) between cryoprotectant type, concentration of cryoprotectant and method of dilution. However, no significant interaction was observed between cryoprotectant type and duration of exposure. Results suggest that cryoprotectant-induced injury to nonfrozen blastocysts is variable and depends on the cryoprotectant used. On the basis of toxicity assays, ethylene glycol was the least harmful and was combined with dimethyl sulfoxide and 1,3-butanediol to produce a new vitrification solution. Mouse blastocysts were successfully cryopreserved using a vitrification solution (designated as VSv) consisting of 20% ethylene glycol, 20% dimethyl sulfoxide and 10% 1,3-butanediol (v/v). Embryos were equilibrated in two steps, first in an equilibration solution (designated as ESv: 10% ethylene glycol, 10% dimethyl sulfoxide and 5% 1,3-butanediol; v/v) and then to VSv or one-step in VSv at different exposure times at room temperature, and then vitrified by direct plunging into liquid nitrogen. High developmental rates were obtained in vitro when the embryos were exposed to ESv and VSv for 3 and 0.5 min, respectively (96.2%) or exposed to VSv for 0.5 min (95.4%). Prolonged exposure time proved detrimental to subsequent embryo development in vitro. When vitrified warmed embryos were transferred immediately to pseudopregnant recipients, the rate of development to normal fetuses did not significantly differ from that of the nonvitrified control (two-step, 54.2 and one-step, 45.0 versus 60.0%, P > 0.05). These results suggest that the simple vitrification solution described in this study is effective for the cryopreservation of mouse blastocysts.     

Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts

Ovine blastocysts were produced by maturation, fertilization and in vitro culture (IVM/IVF/IVC) of oocytes from slaughtered adult and prepubertal ewes and collection from superovulated and inseminated adult animals. Dulbecco's PBS supplemented with 0.3 mM Na Pyruvate and 20% FCS was used as the basic cryopreservation solution. The embryos were exposed to the vitrification solution as follows: 10% glycerol (G) for 5 min, then 10% G +20% ethylene glycol (EG) for 5 min. Embryos were placed into 25% G + 25% EG in the center of 0.25- mL straws and plunged immediately into LN2. Warming was done by placing the straws into a water bath at 37 degrees C for 20 sec, and their contents were expelled into a 0.5 M sucrose solution for 3 min; the embryos were then transferred into 0.25 M and 0.125 M sucrose solution for 3 min each. Warmed blastocysts were transferred to the culture medium for 24 h. Survival was defined as the re-expansion of the blastocoele. All surviving blastocysts were transferred to synchronized recipient ewes, and the pregnancy was allowed to go to term. Of 68 vitrified in vitro produced blastocysts, 46 re-expanded (67.6%) and 10 lambs were born (14.7%). From the 62 in vivo derived and vitrified embryos, 52 re-expanded (83.8%) and 39 lambs were born (62.9%). The lambing rate of in vitro produced fresh transfer embryos was 40% (20 lambs/50 blastocysts transferred), and of the 32 in vivo derived blastocysts and transferred fresh, 26 lambs were born (81.2%). The results indicate that in vitro produced embryos can be successfully cryopreserved by vitrification.     

Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methods

Cryopreservation of equine embryos with conventional slow-cooling procedures has proven challenging. An alternative approach is vitrification, which can minimize chilling injuries by increasing the rates of cooling and warming. The open pulled straw (OPS) and cryoloop have been used for very rapid cooling and warming rates. The objective of this experiment was to compare efficacy of vitrification of embryos in OPS and the cryoloop to conventional slow cool procedures using 0.25 mL straws. Grade 1 or 2 morulae and early blastocysts (< or = 300 microm in diameter) were recovered from mares on Day 6 or 7 post ovulation. Twenty-seven embryos were assigned to three cryopreservation treatments: (1) conventional slow cooling (0.5 degrees C/min) with 1.8 M ethylene glycol (EG) and 0.1 M sucrose, (4) vitrification in OPS in 16.5% EG, 16.5% DMSO and 0.5 M sucrose, or (3) vitrification with a cryoloop in 17.5% EG, 17.5% DMSO, 1 M sucrose and 0.25 microM ficoll. Embryos were evaluated for size and morphological quality (Grade 1 to 4) before freezing, after thawing, and after culture for 20 h. In addition, propidium iodide (PI) and Hoechst 33342 staining were used to assess percent live cells after culture. There were no differences (P > 0.1) in morphological grade or percent live cells among methods. Mean grades for embryos after culture were 2.9 +/- 0.2, 3.1 +/- 0.1, and 3.3 +/- 0.2 for conventional slow cooling, OPS and cryoloop methods, respectively. Embryo grade and percent live cells were correlated, r = 0.66 (P < 0.004). Thus OPS and the cryoloop were similarly effective to conventional slow-cooling procedures for cryopreserving small equine embryos.     

Methanol as a cryoprotectant for equine embryos

Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.     

Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration

The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20–22°C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4°C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22°C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zona dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS + albumin, TCM199 and TCM199 + calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20–22°C, the embryo survival rate decreased (PBS + albumin) or no embryo survived (TCM199 + calf serum) the vitrification procedure. In the third experiment, Day 7 expanded blastocysts were vitrified, thawed, cultured for 1 day, and then re-expanded embryos were again vitrified and thawed. Out of the 87% that survived the first cycle, 73% re-expanded and 47% hatched following the second vitrification and thawing. These observations prove that the vitrification procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages and that an intact zona is not required to obtain high survival rates.     

Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos

Two studies were conducted to evaluate the influence of cryoprotectant, cooling rate, container and cryopreservation procedure on the post-thaw viability of sheep embryos. In Study 1, late morula- to blastocyst-stage embryos were exposed to 1 of 10 cryoprotectant (1.5 M, glycerol vs propylene glycol)-plunge temperature treatments. Embryos were placed in glass ampules and cooled at 1 degrees C/min to -5 degrees C, seeded and further cooled at 0.3 degrees C/min to -15, -20, -25, -30 and -35 degrees C before rapid cooling by direct placement in liquid nitrogen (LN(2)). Post-thaw embryo viability was improved (P<0.01) when embryos were cooled to at least -30 degrees C before LN(2) plunging. Although there were no overt differences in embryo viability between cryoprotectant treatments (each resulted in live offspring after embryo transfer), there was a lower (P<0.01) incidence of zona pellucida damage using propylene glycol (4%) compared to glycerol (40%). In Study 2, embryos were equilibrated in 1.5 M propylene glycol or glycerol or a vitrification solution (VS3a). Embryos treated in propylene glycol or glycerol were divided into ampule or one-step((R)) straw treatments, cooled to -6 degrees C at 1 degrees C/min, seeded, cooled at 0.5 degrees C/min to -35 degrees C, held for 15 minutes and then transferred to LN(2). Embryos vitrified in the highly concentrated VS3a (6.5 M glycerol + 6% bovine serum albumin) were transferred from room air to LN(2) vapor, and then stored in LN(2). Propylene glycol- and glycerol-treated embryos in straws experienced lower (P<0.05) degeneration rates (27%) and yielded more (P<0.05) hatched blastocysts (73 and 60%, respectively) at 48 hours of culture and more (P<0.05) trophoblastic outgrowths (67 and 53%, respectively) after 1 week than vitrified embryos (47, 40 and 20%, respectively). In vitro development rate for VS3a-treated embryos was similar (P>0.10) to that of ampule controls, which had fewer (P<0.05) expanded blastocysts compared to similar straw treatments. Live offspring were produced from embryos cryopreserved by each straw treatment (propylene glycol, 3 of 7; glycerol, 1 of 7; VS3a, 2 of 7). In summary, freeze-preservation of sheep embryos was more effective in one-step straws than glass ampules and propylene glycol tended to be the optimum cryoprotectant. Furthermore, these findings demonstrate, for the first time, the biological competence of sheep embryos cryopreserved using the simple and rapid procedure of vitrification.     

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.     

The ovine uterus as a host for in vitro-produced bovine embryos

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.     

Pregnancies following transfer of equine embryos cryopreserved by vitrification

The objective of this study was to investigate the in vitro and in vivo developmental abilities of equine embryos cryopreserved by vitrification. Twenty-eight embryos were recovered from Native pony and Thoroughbred mares at Days 5 to 7 by nonsurgical uterine flushing (detection of ovulation=Day 0). The vitrification solution contained 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in PBS. The embryos were placed for 1 to 2 min in vitrification solution (Group 1) or following exposure to 20% ethylene glycol in PBS for 10 to 20 min (Groups 2 and 3). Single embryos were loaded in 0.25-ml straws, cooled for 1 min in liquid nitrogen vapor and immersed in liquid nitrogen. Straws were warmed in water (20 degrees C, 20 sec), and the contents were expelled with 0.5 M sucrose in PBS. Then the sucrose was diluted in 1-step (Groups 1 and 2) or 4-steps (Group 3). Embryos (n=21) were cultured for 120 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO(2) in air and evaluated morphologically. Development to the hatching or hatched blastocyst stage was obtained in 0 7 , 4 7 and 4 7 embryos in Groups 1, 2 and 3, respectively. An additional 7 embryos were vitrified-warmed according to the treatment of Group 2 (4 embryos) and Group 3 (3 embryos). Five embryos were selected after in vitro culture for 4 h and were transferred nonsurgically into the uterine horn of Day-4 recipient mares. Transfer of 2 embryos (both Day-6 blastocysts: Group-2 treatment) resulted in pregnancies with a viable fetus at Day-60 of the gestation period.     

Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure

The study was designed to evaluate the efficiency of a modified (sealed) open pulled straw (mOPS) method for cryopreserving rabbit embryos by vitrification or rapid freezing. An additional objective was to determine whether the mOPS method could cause the vitrification of a cryoprotectant solution generally used in rapid freezing procedures. Two consecutive experiments of in vitro and in vivo viability were performed. In Experiment 1, the in vitro viability of rabbit embryos at the morula, compacted morula, early blastocyst and blastocyst stages was assessed after exposure to a mixture of 25% glycerol and 25% ethylene glycol (25GLY:25EG: vitrification solution) or 4.5 M (approximately 25% EG) ethylene glycol and 0.25 M sucrose (25EG:SUC: rapid freezing solution). Embryos were loaded into standard straws or mOPS and plunged directly into liquid nitrogen. The mOPS consisted of standard straws that were heat-pulled, leaving a wide opening for the cotton plug and a narrow one for loading embryos by capillarity. The embryos were aspirated into the mOPS in a column positioned between two columns of cryoprotectant solution separated by air bubbles. The mOPS were then sealed with polyvinyl-alcohol (PVA) sealing powder. The vitrification 25GLY:25EG solution became vitrified both in standard straws and mOPS, whereas the rapid freezing 25EG:SUC solution crystallized in standard straws, but vitrified in mOPS. The total number of embryos cryopreserved was 1695. Embryos cryopreserved after exposure to each solution in mOPS showed higher rates (88.2%) of survival immediately after thawing and removal of the cryoprotectant than those cryopreserved in 0.25 ml standard straws (78.8%; P < 0.0001). After culture, the developmental stage of the cryopreserved embryos significantly affected the rates of development to the expanded blastocyst stage. Regardless of the cryoprotectant used, lower rates of in vitro development were obtained when the embryos were cryopreserved at the morula stage, and higher rates achieved using embryos at blastocyst stages. Based on the results of Experiment 1, the second experiment was performed on blastocysts using the mOPS method. Experiment 2 was designed to evaluate the in vivo viability of cryopreserved rabbit blastocysts loaded into mOPS after exposure to 25GLY:25EG or 25EG:SUC. Embryos cryopreserved in mOPS and 25GLY:25EG solution gave rise to rates of live offspring (51.7%) not significantly different to those achieved using fresh embryos (58.5%). In conclusion, the modified (sealed) OPS method allows vitrification of the cryoprotectant solution at a lower concentration of cryoprotectants than that generally used in vitrification procedures. Rabbit blastocysts cryopreserved using a 25GLY:25EG solution in mOPS showed a similar rate of in vivo development after thawing to that shown by fresh embryos.     

In vitro development following one-step dilution of OPS-vitrified porcine blastocysts

The objective of this experiment was to compare the in vitro survival and hatching rates of OPS-vitrified porcine blastocysts obtained after conventional (three-step dilution) or direct (one-step dilution) warming procedures. Expanded blastocysts were collected by laparotomy from weaned crossbred sows (n=7) on Day 6 of the cycle (D0: onset of estrus). Vitrification was performed as described by Berthelot et al. [Cryobiology 41 (2000) 116] using 17% (v/v) ethylene glycol and 17% (v/v) dimethyl-sulfoxide in the second vitrification medium. Conventional warming was carried out by plunging straws containing embryos in 800 microl of TCM199 Hepes containing 20% new born calf serum (TCM-NBCS) and 0.13 M sucrose for 1 min. Embryos were then transferred to another well with the same medium for 5 min, washed in TCM-NBCS with 0.075 M sucrose for 5 min and transferred to TCM-NBCS for 5 min. In one-step dilution, embryos were placed in 400 microl TCM-NBCS containing 0.13 M sucrose. To evaluate in vitro development, embryos warmed by conventional (n=59) or direct (n=58) procedures were cultured for 96 h. Non-vitrified embryos were used as controls (n=20). No significant (P>0.05) differences were observed in the in vitro development of vitrified and non-vitrified embryos. The survival and hatching rates obtained by three-step dilution (84.8 and 71.2%, respectively) and one-step dilution (86.2 and 74.1%, respectively) procedures were not different (P>0.05). The average diameter of expanded blastocysts from each donor was significantly different (P<0.001) among embryo donors. The embryo diameter or the interactions among the factors evaluated did not affect (P>0.05) the embryo survival and hatching of the vitrified/warmed blastocysts. However, the donor of embryos had a significant effect (P<0.001) on these parameters, confirming previous experiments. This experiment shows that porcine embryo vitrification and one-step dilution are promising procedures to be used under field conditions. However, the good results obtained in vitro must be confirmed also by in vivo experiments.