Factors affecting survival of mouse blastocysts vitrified by a mixture of ethylene glycol and dimethyl sulfoxide

The present study was conducted to determine suitable conditions for mouse blastocysts vitrified by a solution containing 25 % v/v (4.5M) ethylene glycol and 25% v/v (3.4M) dimethyl sulfoxide (VSi). In Experiment 1, blastocysts were exposed to 50% diluted VSi (50% VSi) for 10 minutes then to VSi for 0.5 minutes at room temperature (22 approximately 24 degrees C) or at 4 degrees C, followed by vitrification. The survival rates of these embryos exposed at each temperature were not significantly different. In Experiment 2, embryos were exposed directly to VSi for various time periods at room temperature and diluted in mPBS with 0.5 M sucrose without vitrification. The viability of embryos decreased after more than a 3 minute exposure. When the embryos were exposed to VSi for 0.5, 1, 1.5 and 2 minutes followed by vitrification, the survival rates were 78, 80, 76 and 50%, respectively. In Experiment 3, embryos were vitrified after exposure to 50% VSi for various time periods and then to VSi for 0.5 minutes at room temperature. One minute exposure to 50% VSi resulted in the highest survival rate. In Experiment 4 and 5, the cooling rate (from approximately 70 to approximately 2500 degrees C/minute), sucrose concentration (0, 0.5 and 1 M) of dilution solution, and dilution time (1 or 5 minutes) did not affect the viability of the vitrified embryos. Following exposure to 50% VSi for 1 minute and to VSi for 0.5 minutes at room temperature, embryos were cooled 1) at approximately 2500 degrees C/minute and diluted in 0.5 M sucrose in mPBS after warming or 2) at approximately 200 degrees C/minute and diluted in mPBS. In vivo development rates after the transfer to recipients were 38 and 42%, respectively. These values were similar to that of fresh control embryos.     

The effects of ethylene glycol and dimethyl sulfoxide on cerebroside metastability

Aqueous dispersions of n-acyl cerebrosides are known to exhibit metastable polymorphism of the type: (Formula: see text). The involvement of hydration in this metastable polymorphism has been investigated by differential scanning calorimetric studies of aqueous palmitoylgalactocerebroside (C16:0-CER) dispersions in the presence of agents which disrupt water structure. In the presence of 50 vol% ethylene glycol or 50 vol% dimethyl sulfoxide, only a single reversible ordered----liquid-crystalline transition is observed. This single ordered----liquid-crystalline transition exhibits a smaller enthalpy and occurs at a lower temperature than the major Polymorph II----liquid-crystal transition observed for dispersions in water alone. These results indicate that metastable polymorphism in C16:0-CER is related to hydration.     

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.     

Membrane permeability of the human granulocyte to water,dimethyl sulfoxide,glycerol, propylene glycol and ethylene glycol

Granulocytes are currently transfused as soon as possible after collection because they rapidly deteriorate after being removed from the body. This short shelf life complicates the logistics of granulocyte collection, banking, and safety testing. Cryopreservation has the potential to significantly increase shelf life; however, cryopreservation of granulocytes has proven to be difficult. In this study, we investigate the membrane permeability properties of human granulocytes, with the ultimate goal of using membrane transport modeling to facilitate development of improved cryopreservation methods. We first measured the equilibrium volume of human granulocytes in a range of hypo- and hypertonic solutions and fit the resulting data using a Boyle-van’t Hoff model. This yielded an isotonic cell volume of 378 μm³ and an osmotically inactive volume of 165 μm³. To determine the permeability of the granulocyte membrane to water and cryoprotectant (CPA), cells were injected into well-mixed CPA solution while collecting volume measurements using a Coulter Counter. These experiments were performed at temperatures ranging from 4 to 37 °C for exposure to dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol. The best-fit water permeability was similar in the presence of all of the CPAs, with an average value at 21 °C of 0.18 μm atm⁻¹ min⁻¹. The activation energy for water transport ranged from 41 to 61 kJ/mol. The CPA permeability at 21 °C was 6.4, 1.0, 8.4, and 4.0 μm/min for dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol, respectively, and the activation energy for CPA transport ranged between 59 and 68 kJ/mol.     

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.     

Vitrification of bovine IVF blastocysts in an ethylene glycol/sucrose solution and heat-stable plant-extracted proteins

The use of heat-stable plant proteins in an ethylene glycol-based solution for the vitrification of in vitro-derived embryos was examined. Day 7, 8 and 9 bovine in vitro matured, fertilized and cultured (IVMFC), full and expanded blastocysts were vitrified in solutions composed of 40% ethylene glycol (EG) plus 0.3 M sucrose supplemented with 20% Ficoll and 0.3% BSA (VF-1), 25 mg/ml heat-stable plant proteins (HSPP; VF-2), or with no supplement (VF-3). In Experiment 1, embryos were expelled from the straw after thawing, and EG was diluted from embryos with 0.5 M sucrose. There were no differences in post-thaw embryo survival rates or in hatching/hatched rates after 24 h of culture between the VF-1, VF-2 and VF-3 solutions (40.1, 54.1 and 50.8% and 10.7, 16.4 and 17.5%, respectively). Transfer of 12 frozen/thawed embryos to 6 recipients (2 recipients per treatment) resulted in 2 pregnancies from the VF-2 group and 1 pregnancy from the VF-3 group. In Experiment 2, EG was diluted from embryos after thawing within the straw with 0.5 M sucrose. There were no differences in post-thaw survival or hatching/hatched rates after 24 h of culture (19.0, 13.6 and 23.8% and 9.5, 9.0 and 14.4% for VF-1, VF-2 and VF-3, respectively). Transfer of 6 frozen/thawed embryos to 3 recipients (1 recipient per treatment) resulted in no pregnancies. The post-thaw histology of Day 7, 8 and 9 IVMFC blastocysts showed typical ultrastructure with well preserved cell-to-cell contacts. There were no major differences in the fine structure of blastocysts regardless of treatment. The use of HSPP at a concentration of 25 mg/ml in the vitrification medium did not affect the post-thaw embryo survival over that of no protein supplementation. The presence of macro molecules in a 40% EG/sucrose vitrification solution also did not improve post-thaw viability of IVMFC-derived blastocysts.     

Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient females

Four experiments were conducted to define a system for the direct transfer of frozen-thawed bovine embryos to recipient females. In Experiment I, nonsurgically recovered embryos were frozen in 1.5 M ethylene glycol (EG), 1.5 M propylene glycol (PG), 1.5 M DMSO or 1.4 M glycerol (GLY), and then thawed and placed directly into holding medium. Viability at 72 hours of post-thaw culture was 70, 11, 25 and 30% for the four groups, respectively. In Experiments II and III, 1.0, 1.5 and 2.0 M concentrations of EG were compared; a concentration of 1.5 M appeared to provide optimal cryopreservation and survival after direct rehydration. In Experiment IV, embryos were packaged in straws containing only 1.5 M EG, in straws containing a column of 1.5 M EG and the embryo and two columns of PB1 in a 1:3 ratio of volumes (EG PB1 ), or were frozen in 1.4 M glycerol. After thawing, embryos in EG and EG PB1 treatments were transferred directly to recipient females, while embryos frozen in GLY were rehydrated using a three-step procedure. In the first trial, pregnancy rates at approximately 60 days of gestation for embryos frozen in EG and GLY groups were 39 and 62%, respectively (P<0.10). In the second trial, the pregnancy rate for embryos frozen in EG PB1 was equal to that of embryos frozen in GLY (50% in both groups). These experiments demonstrate the potential for using ethylene glycol as a cryoprotectant for bovine embryos, thus permitting direct transfer of frozen-thawed embryos to recipient females.     

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.     

Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.     

Stimulatory and inhibitory effects of dimethyl sulfoxide and ethylene glycol on ATPase activity and calcium transport of sarcoplasmic membranes.

1. The effect of dimethyl sulfoxide (Me2SO) and ethylene glycol on two different preparations of the sarcoplasmic reticulum, i.e. native membranes and membranes whose phospholipids were hydrolyzed by phospholipase A, were investigated using ATP and p-nitrophenylphosphate as substrates. 2. Me2SO and ethylene glycol inhibit both calcium-dependent ATP hydrolysis and ATP-supported calcium transport by native vesicles. 3. In contrast, calcium-dependent p-nitrophenylphosphatase activity as well as p-nitrophenyl-phosphate-supported calcium transport are activated by both agents at concentrations lower than 30% (v/v). 4. Me2SO strongly stimulates p-nitrophenylphosphate activity of vesicles treated with phospholipase A, but has relatively little effect on p-nitrophenylphosphatase activity of native vesicles. 5. Up to a concentration of approximately 40% Me2SO (v/v) the inhibiting effect on the calcium-dependent ATPase is fully reversible, but only partially reversible on calcium transport. 6. In the concentration range where Me2SO inhibits ATP hydrolysis and calcium transport, it does not affect ATP binding to the membranes nor calcium-dependent formation of phospho-protein. 7. The rate of dephosphorylation as well as the rate of Pi exchange between ATP and ADP are markedly reduced by the presence of 30% Me2SO (v/v). 8. While Me2SO inhibits passive calcium efflux, ethylene glycol produces a considerable activation. 9. ADP-dependent calcium efflux and ATP synthesis are activated by 15% Me2SO (v/v). Ethylene glycol reduces both activities. 10. The results suggest that the respective substrate-enzyme complexes are differently affected by the agents, resulting either in inhibition or stimulation     

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.     

Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.     

Evaluation of the Cryotech Vitrification Kit for bovine embryos

The purpose of this work was to assess commercially available Cryotech Vitrification Kit, in terms of survival, in vitro development and pregnancy rate for bovine embryos. Cumulus-oocyte complexes (COCs) were recovered from ovaries obtained from slaughtered cows and then matured in vitro for 22 h. COCs were fertilized by sex-sorted sperm in IVF-mSOF and cultured in IVC-mSOF for 7 days to the blastocyst stage. Blastocysts were vitrified with the Cryotech Vitrification Kit® and then either warmed to check viability or transferred to synchronized heifers. We observed 100% survival of the in vitro produced blastocysts and obtained the same pregnancy rate (46.8%) as that obtained using fresh in vitro produced blastocysts. We thus conclude that the Cryotech vitrification method is a valid alternative to other vitrification or slow-cooling methods in the bovine species and that it is ready for livestock production.     

Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws

Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene glycol in EFS for 25 to 30 sec at 37 degrees C. Oocytes were loaded into straws in approximately 2 microL of cryoprotectant and plunged directly into LN2. Warming straws and dilution of cryoprotectant was at 37 degrees C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Non-vitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342. Maturation of oocytes to MII after thawing was similar (P>0.05) among groups within species. All equine treatment groups had lower (P<0.01) maturation rates than bovine groups. Live/dead status did not differ among vitrification treatments within species. The percentage of oocytes that survived and reached MII did not differ (P>0.05) within treatment groups of each species. Rates of mature cortical granule distribution did not differ (P>0.05) within species; however, more bovine oocytes (P<0.05) had mature cortical granule distribution and nuclear maturation than equine oocytes. When concurrent cortical granule distribution and nuclear maturation were examined, there was no difference within species; however, only 30% of equine oocytes had nuclear and cytoplasmic maturation compared with 70% of bovine oocytes (P<0.05). In summary, both immature and mature equine and bovine oocytes survived cryopreservation using vitrification in open-pulled straws. However, survival rates were lower for equine than for bovine oocytes.     

Spectroscopic study on structure of horseradish peroxidase in water and dimethyl sulfoxide mixture

The structure of horseradish peroxidase (HRP) in phosphate buffered saline (PBS)/dimethyl sulfoxide (DMSO) mixed solvents at different compositions is investigated by IR, electronic absorption, and fluorescence spectroscopies. The fluorescence spectra and the amide I spectra of ferric HRP [HRP(Fe3+)] show that overall structural changes are relatively small up to 60% DMSO. Although the amide I band of HRP(Fe3+) shows a gradual change in the secondary structure and a decrease in the contents of a helices, its fluorescence spectra indicate that the distance between the heme and Trp173 is almost constant. In contrast, the changes in the positions of the Soret bands for resting HRP(Fe3+) and catalytic intermediates (compounds I and II) and the IR spectra at the C-O stretching vibration mode of carbonyl ferrous HRP [HRP(Fe2+)-CO] show that the microenvironment in the distal heme pocket is altered, even with low DMSO contents. The large reduction of the catalytic activity of HRP even at low DMSO contents can be attributed to the structural transition in the distal heme pocket. In PBS/DMSO mixtures containing more than 70 vol % DMSO, HRP undergoes large structural changes, including a large loss of the secondary structure and a dissociation of the heme from the apoprotein. The presence of the components of the amide I band that can be assigned to strongly hydrogen bonding amide C=O groups at 1616 and 1684 cm(-1) suggests that the denatured HRP may aggregate through strong hydrogen bonds.     

Permeability of intact and dechorionated zebra fish embryos to glycerol and dimethyl sulfoxide

Intact developing embryos of the zebra fish Brachydanio rerio were exposed to [¹⁴C]DMSO and [³H]glycerol (1 M in Fish Ringer) to assess the degree of permeation of these cryoprotectants. Glycerol enters the embryo more easily, although reaching only about 8% of the expected equilibrium level after 2 hr at room temperature; DMSO reaches only about 2.5% of this level. In an attempt to identify the barrier to permeation, dechorionated embryos were similarly exposed to isotopic DMSO. Permeation increased severalfold, indicating that the chorion retards the free exchange of solute. Embryos are unaffected by exposure to 1 M DMSO in Fish Ringer at 23 °C for up to 1 hr. The number of embryos hatching after 1-hr exposure to DMSO at varying concentrations was significantly reduced at 1.5 and 2 M. Embryos exposed to 1 M glycerol for 1 hr at 23 °C showed disruption of periblast cells and separation of the blastoderm; it was impossible to remove glycerol either by abrupt or very slow dilution.     

Cryoprotection by dimethyl sulfoxide and dimethyl sulfone

Preservation of cells and tissues at low temperatures requires the presence of effective cryoprotectants with low toxicity to which cells are relatively permeable. Two similar compounds, dimethyl sulfoxide (DMSO) and dimethyl sulfone (DMSO2), exhibit both features for cryoprotectants, yet DMSO is a very effective cryoprotectant while DMSO2 is ineffective. This anomaly was investigated by relating observations on the phase behavior of DMSO and DMSO2 in aqueous solutions to the recovery of human lymphocytes frozen in the presence of these compounds. The lack of cryoprotection in the presence of DMSO2 appears to be due to the precipitation of DMSO2 from the solution at subzero temperatures. The observation of reduced cell recovery after freezing with increasing concentrations of DMSO2 implies that cell damage is related to the amount of solid DMSO2 present. Precipitation of DMSO2 occurs both intra- and extracellularly, but it is argued that intracellular precipitation of DMSO2 is the damaging phenomenon. Cryoprotective compounds are normally selected based on the criteria of low toxicity and permeability to the plasma membrane. An additional condition, solubility, must be included for interpretation of experimental data and for development of effective protocols for cryopreservation.     

Vitrification of in vitro produced bovine embryos: in vitro and in vivo evaluations

The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based upon in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, ethylene glycol + glycerol (Eg + Gly) + different sucrose concentrations were evaluated. There were no significant differences in development rates among solutions. As for hatching, the Eg + Gly + 0.1 M sucrose group had a greater rate as compared with Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in the evaluations of Day 6, Day 7 and Day 6 + Day 7 embryos; and, Eg + Gly + 0.3 M sucrose group had a greater rate as compared with the Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in evaluations of Day 6 and Day 6 + Day 7 embryos. There were no significant differences in development and hatching rates between Day 6 and 7 in in vitro produced bovine embryos within each treatment group. There were significant differences in nuclei number after vitrification between Eg + Gly + 0.1 M and Eg + Gly + 0 M sucrose groups and the Eg + Gly + 0.5 M sucrose group. Pregnancy after 60 days of transfer and calving rates showed a difference between in vivo produced embryos freshly transferred and in vitro produced embryos vitrified with Eg + Gly + 0.3 M. There were no significant differences in gestation length and sex ratio between treatments. As for birth weight, there were significant differences between fresh in vivo produced embryos and all treatments of in vitro produced embryos. There were significant differences in dystocial parturition between in vivo produced embryos and all treatments with in vitro produced embryos. These results demonstrate that vitrification can be used successfully in the cryopreservation of in vitro produced bovine embryos, and that it might be considered for use in commercial programs.