Direct transfer of bovine embryos frozen-thawed in the presence of propylene glycol or ethylene glycol under on-farm conditions in an integrated embryo transfer program

An integrated bovine embryo transfer program was conducted in collaboration with 11 Japanese prefectural livestock experiment stations. The program was conducted to evaluate the practicability of the direct transfer method for bovine embryos frozen-thawed in the presence of propylene glycol (PG) or ethylene glycol (EG) under on-farm conditions. Embryos at the compacted morula to expanded blastocyst stages were collected from superovulated donors on Day 7 or 8 after estrus and equilibrated in 1.6 M PG or 1.8 M EG in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 20% heat-inactivated calf serum. Embryos were then loaded individually into a 0.25-ml straw and placed directly into a cooling chamber of a programmable freezer precooled to -7 degrees C. After 2 min, the straw was seeded, maintained at -7 degrees C for 8 min more, and then cooled to -30 degrees C either at 0.3 degree C/min or 0.5 degree C/min before being plunged into liquid nitrogen. Embryos at the same stages were also frozen in the presence of 1.4 M glycerol (GLY) by a conventional method, which served as a control. The frozen embryos were thawed by allowing the straws to stand in air for 5 to 10 sec and then immersing them in a 30 degrees C water bath. Embryos frozen-thawed in the presence of PG or EG were nonsurgically transferred into the uterine horn without diluting the cryoprotectant. Embryos frozen-thawed in the presence of GLY were nonsurgically transferred after removing GLY either by the stepwise method (GLY-I) or by in situ dilution with 0.3 M sucrose solution (GLY-II). A total of 1,273 (PG: 400, EG: 418, GLY-I: 177, GLY-II; 278) frozen-thawed embryos was transferred into recipients, yielding 545 pregnancies (overall: 42.8%, PG: 36.0%, EG; 44.7%, GLY-I; 48.6%, GLY-II; 46.0%). The pregnancy rate with PG was significantly lower than that with EG or GLY-II (P < 0.05). The pregnancy rate was affected by the type of cryoprotectant, the region where the embryo transfer program was carried out, the developmental stage of the embryos, the parity of the recipients, and corpus luteum (CL) quality of the recipients. There were no differences in rates of abortion and stillbirth among the 3 cryoprotectants. The present study demonstrates that EG can be effectively used as a cryoprotectant for freezing and direct transfer of bovine embryos, and that the direct transfer method is applicable under on-farm conditions.     

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.     

Direct transfer of equine blastocysts frozen-thawed in the presence of ethylene glycol and sucrose

The present study was designed to examine the suitability of ethylene glycol as a cryoprotectant for equine embryos. Blastocysts recovered nonsurgically from Day 6 donor mares were cryopreserved by conventional 2-step freezing in the presence of 10% ethylene glycol (EG), 10% glycerol (Gly), or 10% ethylene glycol + 0.1M sucrose (EG + Suc). After thawing, the EG and Gly were removed by a 6-step manner, and the EG + Suc was diluted to one fourth in the freezing straw. The postthaw blastocysts were transferred nonsurgically into the uteri of recipient mares on Days 4 to 7 after ovulation. Pregnancy rates, based on Day 15 ultrasonography, were 25.0% (2/8) and 37.5% (3/8) for the blastocysts frozen in EG and Gly, respectively. Direct transfer following thawing and in-straw dilution of blastocysts frozen in EG + Suc resulted in a pregnancy rate of 63.6% (7/11). In fresh Day 6 blastocysts (control group), the pregnancy rate was 70.0% (7/10). These results indicate that the combined use of ethylene glycol and sucrose in a 2-step freezing regimen allows for the direct transfer of frozen-thawed blastocysts into recipient mares, with an acceptable pregnancy rate.     

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.     

New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos

This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.     

Status of cryopreservation of embryos from domestic animals.

The discovery of glycerol as an effective cryoprotectant for spermatozoa led to research on cryopreservation of embryos. The first successful offspring from frozen-thawed embryos were reported in the mouse and later in other laboratory animals. Subsequently, these techniques were applied to domestic animals. Research in cryopreservation techniques have included studies concerning the type and concentration of cryoprotectant, cooling and freezing rates, seeding and plunging temperatures, thawing temperatures and rates, and methods of cryoprotectant removal. To date, successful results based on pregnancy rates have been obtained with cryopreserved cow, sheep, goat, and horse embryos but no success has been reported in swine. Post-thaw embryo survival has been shown to be dependent on the initial embryo quality, developmental stage, and species. The freezing techniques most frequently used in research and by commercial companies are identified as "equilibrium" cryopreservation. In this technique the embryos are placed in a concentrated glycerol solution (1.4 M in PBS supplemented with BSA) at room temperature and the glycerol is allowed to equilibrate for a 20-min period. During the cooling process the straws are seeded (-4 to -7 degrees C) and cooling is continued at a rate of 0.3 to 0.5 degree C/min to -30 degrees C when bovine embryos may be plunged into LN2. Sheep embryos are successfully frozen with ethylene glycol (1.5 M) or DMSO (1.5 M) rather than with glycerol. Horse embryos have been frozen in 0.5 rather than 0.25 cc straws but with cooling rates and seeding and plunging temperatures similar to those used with bovine embryos. Swine embryos have shown a high sensitivity to temperature and cryoprotectants probably due to their high lipid content and a temperature decrease to 15 or 10 degrees C causes a dramatic increase in the percentage of degenerated embryos. However, a recent study has shown that hatched pig blastocysts survived exposure below 15 degrees C. Recent research has shown that embryos may also be frozen by a "nonequilibrium" method. This rapid freezing by vitrification consists of dehydration of the embryo at room temperature by a very highly concentrated vitrification media (3.5 to 4.0 M) and a very rapid freeze that avoids the formation of ice allowing the solution to change from a liquid to a glassy state. Vitrification solutions consist of combinations of sucrose, glycerol, and propylene glycol. With this technique, 50% pregnancy rates have been reported with the bovine blastocyst.     

In vitro survival of murine morulae after quick freezing in the presence of chemically defined macromolecules and different cryoprotectants

We studied the ability of frozen-thawed mouse morulae to develop in vitro when the cryoprotectant proteins were substituted with one of the following nonorganic macromolecules: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and ficoll. We also determined how these agents interacted with 3 different cryoprotectants: glycerol (GLY), propylene glycol (PG), and ethylene glycol (EG). The influence of both of the above factors was measured on the basis of post-thaw morphological appearance, the percentage of development to the expanded blastocyst stage and the total cell count. Morulae (n=950) were collected from superovulated mice. Those classified as good or excellent were distributed among the 12 different freezing solutions, obtained by combining the 3 cryoprotectants with the 4 macromolecules (the 3 mentioned above, plus a control of 5% fetal calf serum) in phosphate buffered saline (PBS). Embryos frozen in PVA, PVP and ficoll tended to be a little difficult to recover from the straws. Development to the expanded blastocyst stage was significantly lower (P<0.05) in propylene glycol (43.6%) than in ethylene glycol (79.5%) or in glycerol (76.1%). Polyvinyl alcohol provided a higher survival rate when combined with glycerol (90.3) or ethylene glycol (95.0), but when it was combined with propylene glycol, only 56.5% of embryos survived after thawing. A positive interaction was observed between glycerol and PVA and between ethylene glycol and PVA or ficoll. The results indicate that fetal serum could be successfully substituted for any of the 3 chemically defined macromolecules. However, our findings also suggest that the use of PG as a cryoprotectant should be avoided when mouse morulae are frozen using the quick freezing method.     

Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

The objectives were to evaluate the reexpansion blastocoele rate, post-thaw viability, and in vitro development of canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol (GLY) or 1.5 m ethylene glycol (EG). Fifty-one in vivo-produced canine blastocysts were randomly allocated in two groups: GLY (n = 26) and EG (n = 25). After thawing, embryos from M0 were immediately stained with the fluorescent probes propidium iodide and Hoechst 33 342 to evaluate cellular viability. Frozen-thawed embryos from M3 and M6 were cultured in SOFaa medium + 10% FCS at 38.5°C under an atmosphere of 5% CO₂ with maximum humidity, for 3 and 6 days, respectively, and similarly stained. The blastocoele reexpansion rate (24 h after in vitro culture) did not differ between GLY (76.5%) and EG (68.8%). Post-thaw viable cells rate were not significantly different between GLY and EG (66.5 ± 4.8 and 57.3 ± 4.8, respectively, mean ± SEM), or among M0 (62.3 ± 5.7%), M3 (56.9 ± 6.0%), and M6 (66.5 ± 6.0%). In conclusion, canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol or 1.5 m ethylene glycol, had satisfactory blastocoele reexpansion rates, similar post-thawing viability, and remained viable for up to 6 days of in vitro culture.     

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

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

Cryopreservation of sheep embryos using ethylene glycol

The objective of the study was to evaluate the use of ethylene glycol (EG) for cryopreservation of sheep embryos. A 2 × 2 factorial treatment arrangement examining one-step vs. two-step cryoprotectant addition and removal was used. The one-step cryoprotectant addition involved placement of embryos directly into 1.5 mol EG, whereas the two-step addition utilized an intermediate 10 min exposure to 0.75 mol EG. Similarly, the one-step cryoprotectant removal involved direct placement of thawed embryos into 1.0 mol sucrose, and the two-step procedure included a 10 min exposure to 0.25 mol sucrose before placement in 1.0 mol sucrose. A total of 185 frozen-thawed embryos was placed into in vitro culture for 96 h to determine viability. No differences were observed between cryoprotectant addition or removal techniques, and overall survival was 69%. To validate the results obtained in vitro, a limited number of embryo transfers was performed. Four ewes receiving a total of 11 frozen-thawed embryos produced eight lambs (73% survival) which compared favorably with 74% survival obtained by transferring 19 non-cryopreserved embryos to eight recipients. It is concluded that one-step addition of 1.5 mol ethylene glycol followed by one-step removal in a 1.0 mol sucrose gradient is an appropriate technique for cryopreservation of sheep embryos.     

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.     

In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycerol

The aim of the present study was to compare the survival rates of goat morulae and blastocysts after different freezing procedures. The viability of frozen-thawed embryos was assessed both in vivo and in vitro. Two cryoprotectants, ethylene glycol and glycerol, were used and three cryoprotectant removal procedures were compared: progressive dilution in 1.0, 0.5, 0.3 and 0 M of cryoprotectant in PBS; a similar progressive dilution with cryoprotectant in PBS plus 0.25 M of sucrose; or one-step transfer in PBS containing 0.25 M of sucrose. In vitro development of frozen-thawed blastocysts was always higher than that of frozen morulae irrespective of the cryoprotectant (52 129 = 40.3% vs 23 161 = 14.3% ; P< 0.001). In vivo, however, frozen-thawed morulae developed equally as well as blastocysts after an identical freezing-thawing protocol. Development both in vivo and in vitro showed ethylene glycol to be a better cryoprotectant than glycerol for goat embryos at both developmental stages (23 vs 0%, 45 vs 35% in vitro; 34.5 vs 21%, 35 vs 23% in vivo for morulae and blastocysts, respectively).     

Vitrification of early-stage bovine and equine embryos

The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25 mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, 7 M ethylene glycol and 0.6 M galactose for 30 s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1 M ethylene glycol and 1.1 M dimethyl sulfoxide (DMSO) for 3 min, 2.5 M ethylene glycol, 2.5 M DMSO and 0.5 M galactose for 30 s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P > 0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P < 0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P < 0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1-3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.     

Surgical recovery and successful surgical transfer of conventionally frozen-thawed embryos in the farmed European polecat (Mustela putorius)

Surgical transfer of in vivo produced conventionally frozen-thawed embryos of farmed European polecat (Mustela putorius) was investigated as a part of an ex-situ preservation program which has the long-term aim of developing a genome resource bank for the endangered European mink (Mustela lutreola). Eighteen oestrous yearling European polecat donors were mated once daily on two consecutive days using 13 fertile males. The donors were surgically flushed for embryos 8-9 days after the first mating. The embryo recovery rate was 60% (116 embryos/193 corpora lutea). The embryos were cryopreserved with 1.5 M ethylene glycol in a programmable freezer using a conventional slow freezing protocol. The thawed embryos were surgically transferred either after dilution with 0.5 M sucrose or directly without removal of ethylene glycol. To induce ovulation, eight recipient females were mated once daily on two consecutive days with vasectomized males starting 7 or 8 days before embryo transfer. The recipients received 7-11 embryos each and three recipients delivered a total of nine pups after a gestation length of 44-46 days. The embryo survival rate was 10% (9 pups/93 frozen embryos). This report describes the first successful cryopreservation of embryos in the Mustelidae family resulting in viable offspring. The low embryo survival rate, however, indicates that the freezing-thawing protocol needs to be improved.     

In vitro comparisons of two cryopreservation techniques for equine embryos: slow-cooling and open pulled straw (OPS) vitrification

Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps with decreasing glycerol and sucrose concentrations. Twenty embryos were vitrified using the OPS method. Embryos were exposed to 7.5% dimethyl-sulfoxide (DMSO)+7.5% ethylene glycol (EG) for 3 min and in 18% DMSO+18% EG+0.4M sucrose for 1 min, loaded in OPS and plunged into liquid nitrogen. After warming, embryos were placed in decreasing sucrose concentrations. All embryos were cultured in synthetic oviduct fluid (SOF) medium for 3h and evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining. The percentage of cells entering in S-phase (%SC) was evaluated by incorporation of BrdU. No significant differences were observed for mean diameter, morphological grade and percentage of degenerate embryos after 3h of culture for slow-cooling and OPS methods. The percentage of dead cells per embryo was similar for the two procedures (42+/-6 versus 46+/-9). The percentage of cells entering in S-phase did not differ significantly between the two procedures (27+/-5 versus 26+/-6). OPS vitrification may be as efficient as slow-cooling for the cryopreservation of equine embryos. However, these results should be confirmed by the transfer of OPS vitrified embryos to recipient mares.     

Effect of different cryoprotectants on the structural preservation of follicles in frozen zebu bovine (Bos indicus) ovarian tissue

Cryopreservation of ovarian tissue is a new and promising technique for germ-line storage. The objective of this study was to evaluate the effect of four cryoprotectants (at two concentrations each) on the preservation of zebu bovine preantral follicles after ovarian cryostorage. Strips of ovarian cortex were cryopreserved using glycerol (GLY; 10 or 20%), ethylene glycol (EG), propanediol (PROH) or dimethylsulphoxide (DMSO; 1.5 or 3M). In addition, a toxicity test was performed for each cryoprotectant by exposing the ovarian tissue to them without freezing. Tissues were analyzed by histology and transmission electron microscopy. Ovarian tissue frozen in either concentration of DMSO or PROH or in 10% GLY retained a higher percentage of morphologically normal follicles (73-88%) than tissue frozen in 20% GLY or in either concentration of EG (16-52%). In the toxicity test, exposure of tissues to DMSO, PROH or GLY resulted in higher percentages of normal follicles (80-97%) than exposure to EG (49%). Electron microscopy revealed damage to the ultrastructure of follicles frozen in 10% GLY, while follicles cryopreserved in DMSO and PROH at either concentration exhibited normal ultrastructure. In conclusion, DMSO and PROH were the most effective cryoprotectants for zebu ovarian tissue, preserving the structural integrity of somatic and reproductive cells within the ovary.     

Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos

Experiments were conducted to determine viability of equine embryos in vivo after vitrification. In a preliminary study (Experiment 1), embryos were exposed in three steps to vitrification solutions containing increasing concentrations of ethylene glycol and glycerol (EG/G); the final vitrification solution was 3.4 M glycerol + 4.6 M ethylene glycol in a base medium of phosphate-buffered saline. Embryos were warmed in a two-step dilution and transferred into uteri of recipients. No pregnancies were observed after transfer of blastocysts >300 microm (n = 3). Transfer of morulae or blastocysts < or = 300 microm resulted in four embryonic vesicles (4/6, 67%). In a second experiment, embryo recovery per ovulation was similar for collections on Day 6(28/36, 78%) versus Days 7 and 8(30/48, 62%). Embryos < or = 300 and >300 microm were vitrified, thawed and transferred as in Experiment 1. Some embryos < or = 300 microm were also transferred using a direct-transfer procedure (DT). Embryo development rates to Day 16 were not different for embryos < or = 300 microm that were treated as in Experiment 1(10/22, 46%) or transferred by DT (16/26, 62%). Embryos > 300 microm (n = 19) did not produce embryonic vesicles.     

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.     

Successful transfer of vitrified Ilama (Lama glama) embryos

The exploitation of the domestic animals species of South American camelids is of great social importance for the native people living in the High Andes. The reproductive physiology of these species is a unique challenge in the development of advanced breeding techniques. At present, the cryopreservation of embryos has not been developed and very few investigations have been conducted. The objective of the present work was to evaluate the in vivo survival of vitrified llama embryos after transfer to recipient females. Donors females were treated with a CIDR-estradiol benzoate-eCG regimen and were mated naturally 6 days after CIDR withdrawal. One ovulatory dose (8 microg) of GnRH was administered immediately after mating. A second mating was allowed 24 h later. Embryo recovery was performed nonsurgically between 8 and 8.5 days after the first mating. Twenty-two ova/embryos were recovered from 12 donor females. Hatched blastocysts were exposed to vitrification solution (20% glycerol + 20% ethylene glycol + 0.3 M sucrose + 0.375 M glucose + 3% polyethylene glycol (P/V)) in three steps, and after loading into 0.25 ml straws, were plunged into liquid nitrogen. For embryo transfer, recipients animals were ovulation-synchronized using GnRH administered at the same time as donors. A total of eight vitrified-warmed embryos and 12 fresh embryos were nonsurgically transferred to four and six recipient females, respectively (two embryo per recipient). The pregnancy rates were 50 and 33.3% for recipients that had received vitrified embryos and fresh embryos, respectively. The results demonstrated the effectiveness of this simple vitrification method for cryopreservation of llama embryos.     

Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide

The influence of equilibration time before vitrification on the viability of vitrified morula- to blastocyst-stage bovine embryos and in vivo viability of vitrified embryos following transfer to recipients were investigated. In experiment 1, the embryos were exposed to an equilibration solution (50% VSED) containing 12.5% v/v ethylene glycol and 12.5% v/v dimethyl sulfoxide in modified Dulbecco's phosphate buffered saline with 4 mg/ml BSA (m-PBS) for 1, 2 and 5 minutes at room temperature (22 to 24 degrees C). The embryos were then placed in 15mul vitrification solution (VSED) consisting of 25% v/v ethylene glycol and 25% v/v dimethyl sulfoxide in m-PBS and were loaded into 0.25 ml plastic straws at room temperature. After 30 seconds, the straws were placed in liquid nitrogen (LN(2)) vapor for 2 minutes, plunged and stored in LN(2). To thaw, the straws were warmed in water at 20 degrees C for 15 seconds and the contents of the straws were expelled into a plastic dish. The embryos were diluted in 0.5 M sucrose + m-PBS for 5 minutes and were cultured in TCM-199 supplemented with bovine oviductal epithelial tissue. Viability of the embryos was assessed by the forming or reforming of the blastocoele after 24 hours of culture. High in vitro survival rates (73 approximately 90%) of vitrified embryos were obtained after 1 and 2 minute equilibrations, but was reduced (P<0.05) after 5 minute equilibration. In Experiment 2, morula- to blastocyst-stage embryos were vitrified after 1 minute equilibration in 50% VSED and 30 seconds of exposure to VSED. The vitrified-warmed embryos were transferred to recipient heifers at 7 days after estrus (1 embryo per recipient). Five (38%) of 13 (40%) of 10 recipients that had received blastocysts were diagnosed as pregnant using ultrasonography 60 days following transfer.