Vitrification of mouse oocytes in ethylene glycol-raffinose solution: effects of preexposure to ethylene glycol or raffinose on oocyte viability.

We investigated the effects of preexposure to ethylene glycol (EG) or raffinose on the viability of vitrified mouse oocytes. Ovulated oocytes at the metaphase II stage were preexposed either to 2 M EG for 0, 2, or 5 min or to ascending concentrations (0.15 followed by 0.3 M ) of raffinose solution for 2, 5, or 10 min each (here referred to as 2-2, 5-5, and 10-10 min, respectively). The oocytes were then exposed to a vitrification solution (VS), 6 M EG + 0.3 M raffinose, for 0.5, 1, 2, or 5 min and then vitrified or immediately diluted. After warming, the developmental capacity of oocytes was determined after in vitro fertilization. Volume changes in oocytes during preexposures and exposure to the VS were also investigated. The results demonstrated that preexposure to 2 M EG allowed shorter exposure times of oocytes to the VS and that predehydration in raffinose solutions for 5-5, but not 2-2 or 10-10 min, allowed a wider range of exposure times to the VS. Experiments on volume change suggested that the optimum time of exposure to the VS depends on the amount of EG permeation after preexposure to 2 M EG or to raffinose solutions. Preexposures to 2 M EG or raffinose under optimized conditions increased the viability of vitrified-warmed oocytes compared to direct exposure to VS without preexposures.     

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.     

Optimization of triacetate cellulose hollow fiber vitrification (HFV) method for cryopreservation of in vitro matured bovine oocytes

The aim of the current work was to evaluate applicability of triacetate cellulose hollow fiber vitrification (HFV) method for cryopreservation of groups of in vitro matured bovine oocytes (12–17 oocytes per device). We also attempted to optimize HFV protocol by altering concentration of non-permeating cryoprotectant (sucrose) in vitrification solution and concentration of extracellular Ca²⁺ by using a calcium-free base medium for preparation of vitrification/rewarming solutions with ethylene glycol (EG) as a single permeating cryoprotectant. Neither of modifications of HFV protocol significantly affected survival or fertilization rates of the vitrified bovine oocytes. Embryo development rates in the vitrification groups were lower than those in the control (31.2% of blastocysts at Day 8 post IVF). Use of vitrification/rewarming solutions with lower Ca²⁺ concentration and EG did not significantly improve embryo development rates. An increase of sucrose concentration in vitrification solution from 0.5 to 1.0 M significantly improved blastocyst yield on Day 8 post IVF (21.1–23.4% vs 3.1–3.5%; p < 0.05). Obtained results indicated that sufficient dehydration of the oocytes and/or the solution surrounding them in hollow fiber before immersion into liquid nitrogen is an important factor for successful vitrification. Use of HFV method allowed simplification and standardization of vitrification/rewarming procedures. Triacetate cellulose hollow fibers can be used successfully for cryopeservation of groups of in vitro matured bovine oocytes.     

Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.     

Effects of six sugars on the longevity,fecundity and nutrient reserves of Microplitis mediator

Parasitoid adults can directly feed on floral nectar and honeydew containing monosaccharides and disaccharides. Oligosaccharides such as maltose, melezitose and raffinose are also found in honeydew but are rare in floral nectar. The effects of six different sugar resources on the longevity, fecundity and nutrient reserves of Microplitis mediator, a larval endoparasitoid in the cotton bollworm Helicoverpa armigera (Hübner) were determined in our laboratory. The results showed that both food and sex affected longevity of this wasp. Females and males of M. mediator fed with 1 M sucrose solution survived longer than controls fed with water (5.7- and 3.7-fold longer, respectively). When provided with sucrose, glucose or fructose solutions, the parasitoid generated 3.6- to 3.7-fold more offspring than controls, and 60–75% of these progenies were produced during the first 5 days. When separately given fructose, sucrose or glucose, this wasp accumulated fructose and total sugar at the highest level, which means a high sugar levels might lead to prolonging longevity and more offspring in M. mediator. In addition, compared with organisms fed galactose or raffinose, M. mediator fed sucrose or fructose accumulated high glycogen levels. Furthermore, in M. mediator, the lipid content declined with the advancing age. Females showed the slowest lipid metabolic rates when fed with sucrose, glucose, fructose, mannose and galactose solutions versus when fed with raffinose and control. In addition, only sucrose had a significant effect on lipid levels in males nearing the end of life.     

Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation.

Solutions used for vitrification or rapid cooling of embryos usually contain high concentrations of penetrating cryoprotectants. At these concentrations embryos can tolerate the penetrating cryoprotectants for only short periods of time without damage. This study designed and tested cryoprotectant solutions that combined high polymer concentrations with low penetrating cryoprotectant concentrations. Mouse 2-cell embryo development was not compromised by up to 15-min exposure to 30 wt% solutions of the polymers Ficoll 70,000 MW or dextran 69,000 MW at room temperature. However, our batches of polyvinylpyrrolidone (PVP) 10,000 and PVP 40,000 were embryo-toxic even after extensive dialysis against Milli-Q water. As both Ficoll and dextran contribute to a solution's physical vitrification properties, we formulated vitrifying solutions containing only 11 to 27 wt% ethylene glycol (EG) by including 34 to 49 wt% polymers (27 wt% EG + 34 wt% Ficoll, 27 wt% EG + 34 wt% dextran, 16 wt% EG + 39 wt% Ficoll, or 11 wt% EG + 49 wt% Ficoll, in phosphate-buffered saline (PBS)). Novel solutions were designed for 0.25 ml straw as a viscous matrix for encapsulation of embryos. These yielded high rates of development of 2-cell mouse embryos after rapid cooling and warming (> or = 96% expanded blastocysts in vitro and > or = 62% viable fetuses as assessed on day 15 of gestation in vivo) in all tested solutions. All control 2-cell embryos formed expanded blastocysts in vitro and 78% formed fetuses in vivo. Comparable results were obtained with both 4-cell and 8- to 16-cell mouse embryos. The lower toxicity of Ficoll and dextran may explain why these new solutions gave better results than had previously been reported for solutions containing 7.5% PVP and low concentrations of EG (2 M).     

Gustatory perception and metabolic utilization of sugars by<Emphasis Type="Italic"> Myrmica rubra</Emphasis> ant workers

The suitability of various nectar and honeydew sugars as a food source for the polyphagous ant species M. rubra (L.) was studied. The sugars used included monosaccharides (fructose, glucose, galactose, mannose, rhamnose), disaccharides (sucrose, maltose, trehalose, melibiose, lactose) and trisaccharides (melizitose, raffinose, erlose). Single-sugar solutions were tested on ant workers in a long-term laboratory bioassay in which acceptance of the solutions and ant survival were recorded. The acceptance of the sugars was confirmed in a second bioassay in which feeding time was established. Enzymatic hydrolysis of sucrose, maltose and melibiose was investigated through HPLC analyses of workers fed these disaccharides. Sugar acceptance and feeding time were related to ant survival. Considering the monosaccharide units of which the sugars are composed, fructose seems especially suitable as a short-term energy source, while glucose appears to be used both directly and for storage. The presence of a galactose unit appears to reduce sugar suitability. It is suggested that the workers possess invertase and maltase and to a lesser degree also galactosidase. The gustatory perception is correlated with the profitability of sugars in further metabolic processes.     

Post-vitrification survival and in vitro maturation rate of buffalo (Bubalus bubalis) oocytes: effect of ethylene glycol concentration and exposure time

The present study was undertaken to investigate the effects of ethylene glycol concentration and time of exposure to equilibration solution on the post-thaw morphological appearance and the in vitro maturation rate of buffalo oocytes. Vitrification solution-I (VS-I) consisted of 4.5M ethylene glycol (EG), 3.4M dimethyl sulphoxide, 5. 56mM glucose, 0.33mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), whereas vitrification solution-II (VS-II) contained 3.5M EG, with other constituents at same concentrations as in VS-I. The equilibration solutions-I and II were prepared by 50% dilution (v/v) of VS-I and VS-II, respectively, in DPBS. Prior to vitrification, the cumulus-oocyte complexes (COCs) were exposed to equilibration solution-I or II for 1 or 3min at room temperature (25-30 degrees C). Groups of four to five oocytes were then placed in 15microl of respective vitrification solution, and immediately loaded into 0. 25ml French straws, each containing 150microl of 0.5M sucrose in DPBS. The straws were placed in liquid nitrogen (LN(2)) vapour for 2min, plunged and stored in LN(2) for at least 7 days. The straws were thawed by keeping in warm water at 28 degrees C for 20s, and the oocytes were equilibrated for 5min in 0.5M sucrose for one-step dilution. The percentage of oocytes found to be morphologically normal varied from 89 to 96% for the two equilibration solutions and the two exposure times. Among the damaged oocytes, cracking of zona pellucida was the abnormality observed most frequently. The nuclear maturation rate of oocytes equilibrated in equilibration solutions-I and II for 1 (28 and 24%, respectively) or 3min (32 and 33%, respectively) did not differ significantly. These results show that it is possible to cryopreserve buffalo oocytes by vitrification using a combination of 3.5M EG and 3.4M DMSO with an exposure time of 3min.     

Vitrification of in vitro cultured rabbit morulae

In the present work, we attempt to establish an efficient vitrification procedure for 32-cell rabbit embryos obtained in vitro. In experiment 1, both the effect of the composition of the vitrification solutions and the cryoprotectant addition (either in one or two steps) were studied. For one-step addition, straws with embryos in the final vitrification solution (total time 60s) were plunged into liquid nitrogen. For two-step addition, previously embryos were 2 min pre-equilibrated in 0.5 ml of (1:1) PBS plus 20% FCS: vitrification solution without sucrose. Different solutions of cryoprotectants were compared: 25 vol.% ethylene glycol supplemented with 0.25 M sucrose (25EG+S) and 20% ethylene glycol plus 20% dimethyl sulfoxide, alone (20EG+20DMSO-S) or supplemented with 0.25 M sucrose (20EG+20DMSO+S). Six percent (30/487) of the total of 32-cell embryos obtained by in vitro culture in each experimental session was slow-frozen by a classical method as a technical efficiency control. Only 30% slow-frozen embryos reached blastocyst stage. Significant differences in embryo development were detected between the one-step (25EG+S) and two-step (25EG+S) groups and the one-step (20EG-20DMSO+S) and two-step (20EG-20DMSO-S) groups (0-6% versus 36-50%, respectively). Consequently, in the following experiments only these two vitrification procedures were used. In experiment 2, we attempted to substitute the use of PBS by HEPES-buffered Ham's F-10 (h-CM) in all cryoprotective solutions or media. When h-CM was used, a significant reduction in the in vitro embryo development was observed when the HEPES-buffered groups were compared with one-step (20EG-20DMSO+S) group in s-PBS (35-45% versus 73%). In experiment 3, the one-step (20EG+20DMSO+S) and two-step (20EG+20DMSO-S) procedures were assayed using two FCS levels (20 and 40%) in the PBS-based media. Relative to in vitro development, the highest rates were reached with one step (20EG-20DMSO+S), using PBS plus 20% FCS, which was different from two steps (20EG-20DMSO-S), regardless of percentage of FCS in the PBS-based media (81% versus 41-45%; P<0.05). In conclusion, we propose either the one step (20EG-20DMSO+S) or two steps (20EG-20DMSO-S) prepared in PBS plus 20% serum for use in future works.     

Vitrification of pronuclear-stage mouse embryos on solid surface (SSV) versus in cryotube: comparison of the effect of equilibration time and different sugars in the vitrification solution

The cryopreservation of pronuclear-stage embryos has particular importance in transgenic technology and human assisted reproductive technology (ART). The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage mouse embryos. Two vitrification methods (solid surface vitrification (SSV) vs. vitrification in cryotube) have been compared with special emphasis on the effect of the exposure of the embryos to the solutions for various times and the sugar content (trehalose, sucrose, or raffinose) of the vitrification solutions. Pronuclear-stage embryos were either exposed to 1 M dimethyl sulfoxide (DMSO) + 1 M propylene-glycol (PG) solution for 2, 5, 10, or 15 min or not exposed to this "equilibration" solution. The vitrification solutions consisted of 2.75 M DMSO and 2.75 M PG in M2 medium supplemented with 1 M trehalose (DPT), 1 M sucrose (DPS), or 1 M raffinose (DPR). In the cryotube method, groups of 15-25 embryos were transferred into a 1.8 ml cryotube containing 30 microl of DPT, DPS, or DPR. After 30 sec, the cryotubes were directly plunged into liquid nitrogen (LN(2)) and stored for 1 day to 1 month. Vitrified samples were warmed by immersing the cryotubes in a 40 degrees C water bath and then immediately diluted with 300 microl of 0.3 M trehalose, sucrose, or raffinose in M2. In the SSV method, after equilibration 15-20 embryos were exposed to DPT, DPS, or DPR solutions for around 20 sec before being dropped in 2-microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. Vitrified droplets were stored in cryovials in LN(2). Warming was performed by transferring the vitrified droplets into 0.3 M solutions of trehalose, sucrose, or raffinose at 37 degrees C, respectively. Results showed that both SSV and cryotube vitrification methods can result in high rates of in vitro blastocyst development (up to 58.3 and 68.5% with DPR, respectively), not statistically different from that of the controls (58.3 and 64.4%). Even without the equilibration step prior to vitrification, relatively high-survival rates have been achieved, except for the DPS solution. In conclusion, vitrification of pronuclear-stage mouse embryos can result in high rates of in vitro development to blastocyst, and the use of raffinose in the vitrification solution is advantageous to improve cryosurvival.     

Effect of different cryoprotectants and vitrificant solutions on the hatching rate of turbot embryos (Scophthalmus maximus)

Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, in order to achieve a vitrifiable medium, a high concentration of permeable cryoprotectants must be employed, and the incorporation of high molecular weight compounds should also be considered. The toxicity of these permeable and non-permeable agents has to be assessed, particularly when high concentrations are required. In the present study, permeable and non-permeable cryoprotectant toxicity was determined in turbot embryos at two development stages (F stage-tail bud and G stage-tail bud free). Embryos treated with pronase (2mg/ml, 10 min at 22 degrees C) were incubated in dimethyl sulfoxide (Me2SO), methanol (Meth.) or ethylene glycol (EG) in concentrations ranging from 0.5 to 6M for periods of 10 or 30 min, and in 5, 10, and 15% polyvinylpyrrolidone (PVP), 10, 15, and 20% sucrose or 0.1, 1, and 2% X-1000 for 2 min. The embryos were then washed well and incubated in seawater until hatching. The toxicity of permeable cryoprotectants increased with concentration and exposure time. There were no significant differences between permeable cryoprotectants. However, embryos tolerated higher concentrations of Me2SO than other cryoprotectants. Exposure to permeable cryoprotectants did not affect the hatching rate except at G stage with X-1000 treatment and 20% sucrose. Taking into account the cryoprotectant toxicity and the vitrification ability of cryoprotectant mixtures, three vitrification solutions (V1, V2, and V3), and one protocol for stepwise incorporation were designed. The tested solutions contained 5M Me2SO+2M Meth+1M EG plus 5% PVP, 10% sucrose or 2% X-1000. The hatching rate of embryos that had been exposed to the the vitrification solutions was analyzed and no significant differences were noticed compared with the controls. Our results demonstrate that turbot embryos can be subject to this cryoprotectant protocol without deleterious effect on the hatching rate.     

Immature bovine oocyte cryopreservation: comparison of different associations with ethylene glycol, glycerol and dimethylsulfoxide

Research on different cryoprotectants and their associations is important for successful vitrification, since greater cryoprotectant concentration of vitrification solution may be toxic to oocytes. The aim of the present research was to compare the efficiency of immature bovine oocyte vitrification in different associations of ethylene glycol (EG), glycerol and dimethylsulfoxide (Me(2)SO). In the first experiment, oocytes were exposed to the cryoprotectant for either 30 or 60s in final solutions of EG+DMSO1 (20% EG+20% Me(2)SO) or EG+DMSO2 (25% EG+25% Me(2)SO) or EG+GLY (25% EG+25% glycerol). In the second experiment, the oocytes were vitrified in open pulled straws (OPS) using 30s exposure of final solutions of EG+DMSO1 or EG+DMSO2 or EG+GLY. Maturation rates of 30s exposure groups were not different from the control, but 60s cryoprotectant exposure was toxic, decreasing maturation rates. The vitrification with EG+DMSO2 resulted in enhanced maturation rate (29.2%) as compared with EG+DMSO1 (11.7%) and EG+GLY (4.3%) treatments. These data demonstrate that concentration and type of cryoprotectant have important effects on the developmental competence of vitrified oocytes.     

Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80-93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.     

Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived

We compare different vitrification protocols on the pregnancy and lambing rate of in vitro produced (IVP) and in vivo derived (IVD) ovine embryos. Ovine blastocysts were produced by in vitro maturation, fertilization and culture of oocytes collected from slaughtered ewes or superovulated and inseminated animals. Embryos were cryopreserved after exposure at room temperature either for 5 min in 10% glycerol (G), then for 5 min in 10% G + 20% ethylene glycol (EG), then for 30 s in 25% G + 25% EG (glycerol group), or for 3 min in 10% EG + 10% dimethyl sulphoxide (DMSO), then for 30s in 20% EG + 20% DMSO + 0.3 M sucrose (DMSO group). One group of in vitro produced embryos was cryopreserved similarly to the DMSO group, but with 0.75 M sucrose added to the vitrification solution (DMSO 0.75 group). Glycerol group embryos were then loaded into French straws or open pulled Straws (OPS) while the DMSO group embryos were all loaded into OPS and directly plunged into liquid nitrogen. Embryos were warmed with either a one step or three step process. In the one step process, embryos were placed in 0.5 M sucrose. The three-step process was a serial dilution in 0.5, 0.25 and 0.125 M sucrose. The embryos of DMSO 0.75 group were warmed directly by plunging them into tissue culture medium-199 (TCM-199) + 20% foetal bovine serum (FBS) in the absence of sucrose (direct dilution). Following these manipulations, the embryos were transferred in pairs into synchronised recipient ewes and allowed to go to term. The pregnancy and the lambing rate within each group of IVP and IVD embryos indicated that there was no statistical difference among the vitrification protocols.     

Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest

Phase diagrams are of great utility in cryobiology, especially, those consisting of a cryoprotective agent (CPA) dissolved in a physiological salt solution. These ternary phase diagrams consist of plots of the freezing points of increasing concentrations of solutions of cryoprotective agents (CPA) plus NaCl. Because they are time-consuming to generate, ternary diagrams are only available for a small number of CPAs. We wanted to determine whether accurate ternary phase diagrams could be synthesized by adding together the freezing point depressions of binary solutions of CPA/water and NaCl/water which match the corresponding solute molality concentrations in the ternary solution. We begin with a low concentration of a solution of CPA+salt of given R (CPA/salt) weight ratio. Ice formation in that solution is mimicked by withdrawing water from it which increases the concentrations of both the CPA and the NaCl. We compute the individual solute concentrations, determine their freezing points from published binary phase diagrams, and sum the freezing points. These yield the synthesized ternary phase diagram for a solution of given R. They were compared with published experimental ternary phase diagrams for glycerol, dimethyl sulfoxide (DMSO), sucrose, and ethylene glycol (EG) plus NaCl in water. For the first three, the synthesized and experimental phase diagrams agreed closely, with some divergence occurring as wt% concentrations exceeded 30% for DMSO and 55% for glycerol, and sucrose. However, in the case of EG there were substantial differences over nearly the entire range of concentrations which we attribute to systematic errors in the experimental EG data. New experimental EG work will be required to resolve this issue.     

Development of vitrified bovine secondary and primordial follicles in xenografts

The objective was to evaluate the effect of various vitrification conditions on the morphology of bovine secondary and primordial follicles, and to use xenografting to confirm their developmental ability. Secondary follicles were placed in vitrification solution containing 15% (v:v) ethylene glycol (EG), 15% (v:v) dimethyl sulfoxide (DMSO), 20% (v:v) fetal calf serum (FCS), and 0, 0.25, or 0.5 M sucrose at room temperature for 1 or 30 min, or at 4 °C for 30 min before being plunged into liquid nitrogen (LN₂). Ovarian tissues with primordial follicles were equilibrated in a solution containing 7.5% EG, 7.5% DMSO, and 20% FCS for 5 or 15 min, and then treated with a vitrification solution (15% EG, 15% DMSO, and 20% FCS) containing 0 or 0.5 M sucrose at room temperature for 1 min, and then plunged into LN₂. One week later, follicles and tissues were warmed, and morphology assessed histologically. Secondary follicles vitrified in sucrose-free solution had more oocytes with shrinkage of the nucleus and abnormal cytoplasm relative to those vitrified in sucrose-containing solution. When primordial follicles were equilibrated for 5 min and vitrified in sucrose-free solution, the percentage of morphologically normal primordial follicles was higher than in the other groups (P < 0.05). After 4 wk and 6 mo of xenografting of vitrified-warmed secondary and primordial follicles, respectively, in SCID mice, follicles developed to the antral stage and oocytes grew. In conclusion, bovine secondary follicles were successfully cryopreserved in sucrose-containing vitrification solutions and maintained their ability to develop to the antral stage and grow oocytes, whereas primordial follicles vitrified in sucrose-free solution maintained their morphology and developed to the antral stage, with oocyte growth.     

On the assessment of the stability of vitrified cryo-media by differential scanning calorimetry: A new tool for biobanks to derive standard operating procedures for storage,access and transport

When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me₂SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4 wt% changed the devitrification time with a factor of 342 and 271 for Me₂SO and EG, respectively. Concentration changes in EG/Me₂SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.     

Vitrification of domestic cat (Felis catus) ovarian tissue: Effects of three different sugars

We aimed to evaluate the effect of three extracellular cryoprotectants on the morphology of vitrified feline preantral follicles. Feline ovarian fragments (0.5 × 2 × 2 mm) collected from five domestic adult cats subjected to ovariohysterectomy for routine castration were vitrified with ethylene glycol (EG) 40% combined or not with sucrose (0.1 or 0.5 M), trehalose (0.1 or 0.5 M), or raffinose (0.1 M). After vitrification using the solid-surface method and warming of the tissues, cryoprotectants were washed out of the ovarian tissues, which were fixed for histological analysis. The percentages of normal follicles were similar to the control (fresh) (62.9 ± 4.1%) only for tissues exposed and cryopreserved with EG + trehalose at concentrations of 0.1 (35.8 ± 8.3%) and 0.5 M (33.4 ± 5.4%). All the other sugars decreased the percentages of morphologically normal follicles as compared to the control group and the trehalose groups. Based on the results of the present study, we recommend the use of trehalose as the extracellular cryoprotectant for the vitrification of feline ovarian tissue.     

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.