Vitrification Protocol Research Articles

Successful pregnancies and deliveries after a simple vitrification protocol for day 3 human embryos

Objective: To evaluate the efficacy and efficiency of freezing cleaved human embryos through vitrification.Design: Clinical study of vitrification of human embryos.Setting: Assisted reproductive technology centers.Patient(s): Thirty-six patients undergoing IVF-ICSI treatment whose surplus embryos were frozen.Intervention(s): Two hundred fifteen surplus embryos vitrified, subsequently thawed, and transferred in natural or controlled cycles.Main Outcome Measure(s): Embryo survival rate after thawing and resultant patient pregnancy rate.Result(s): From the 215 vitrified and thawed embryos, 106 survived, with an overall embryo survival rate of 49.3%. The survival rate was higher when embryos were vitrified at the eight-cell stage compared with at the six to seven–cell and six-cell stages (79.2%, 39.7%, and 21.1%, respectively). On average, 2.9 ± 1.2 embryos per patient were transferred, resulting in 11 pregnancies (30.5%), with an implantation rate of 10.4% per embryo transferred.Conclusion(s): Ultrarapid embryo freezing by vitrification of eight-cell stage embryos is a reliable method, as evidenced by high rates of embryo survival and pregnancy, making it a superior alternative to the conventional slow-cooling method.

Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrification of dormant shoot tips

Shoot tips excised from dormant axillary buds of persimmon (Diospyros kaki Thunb.) were cryopreserved by vitrification. These excised shoot tips were dehydrated in a highly concentrated vitrification solution for 20min at 25°C and then plunged directly into liquid nitrogen. After rapid warming in water at 40°C, the shoot tips were rinsed in a 1.2M sucrose solution for 20min and then plated on a solidified culture medium. Successfully vitrified shoot tips resumed growth within 10days of plating and developed shoots within 3weeks without intermediary callus formation. This simple protocol was successfully applied to the 16 cultivars found in the temperate zone. The average rate of shoot formation was 89%. Even the subtropical species of Diospyros demonstrated a very high recovery growth when the shoot tips had been previously osmoprotected with a mixture of 2M glycerol plus 0.4M sucrose for 20min following sucrose preculture. Little or no contamination occurred in the cryopreserved shoot tips excised from sterilized winter axillary buds. Thus, this simple and reliable vitrification protocol using dormant shoot tips appears to be promising as a routine method for the long-term conservation of Diospyros germplasm of both temperate and subtropical origins.

Cryopreservation of Shoot Tips from Six Endangered Australian Species using a Modified Vitrification Protocol

An efficient vitrification procedure was developed and successfully applied to cryopreserve six endangered West Australian species (family Haemodoraceae: Anigozanthos humilis ssp. chrysanthus Hopper;A. kalbarriensis Hopper;A. viridis ssp. terraspectans Hopper;Conostylis dielsia ssp.teres Hopper;C. micrantha Hopper and C. wonganensis Hopper). Species were initially evaluated for cryostorage using a basic vitrification protocol involving: culturing plantlets in vitro for 21d; excision of shoot apices; preculture of apical tips on 0.4M sorbitol for 2d, followed by incubation in PVS2 (plant vitrification solution 2) for 25min at 0°C, then direct immersion in liquid nitrogen (LN). Warming of retrieved material was for 1min in a 40°C water bath. Using this protocol five of the six species exhibited low post-storage survival, while the sixth species, A. viridis ssp. terraspectans posted higher survival (61.1%). Using A. viridis ssp. terraspectans as an indicator species, the initial protocol was modified to include: 3d preculture on 0.80M glycerol, loading treatment with 2.0M glycerol plus 0.4M sucrose solution for 20min, followed by 25min exposure to a modified PVS2. Survival was significantly improved in the test species, and in further experiments three other species also showed significant improvements with the new protocol. Key findings include: effectiveness of glycerol in the preculture medium; the effect of preculture duration; the importance of a loading stage for these species; and the successful use of modified PVS2 solutions with reduced or zero dimethyl sulfoxide (DMSO).

Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation

The efficacy of several sugars and polyalcohols in preculture medium was investigated using Anigozanthos viridis ssp terraspectans Hopper (Haemodoraceae), a threatened plant species endemic to the south west of Western Australia. A vitrification protocol involving preculturing of shoot apices for 3 days on different concentrations of sugars and polyalcohols, followed by incubation in plant vitrification solution 2 (PVS2) for 25 min, prior to immersion in liquid nitrogen (LN) and warming resulted in shoot tip survival ranging from 34 to 84%. High levels of survival were obtained with polyalcohols, compared to sucrose, glucose, trehalose and raffinose when used at the same molarity (0.4 M) or at the equivalent concentration of total hydroxyl (OH) groups present in molecules. In both cases glycerol proved more effective. When polyalcohols (ribitol and erythritol) with similar stereochemical arrangement of OH groups as glycerol were examined, at the same molarity (0.4 M) and with equivalent OH numbers, higher survival was achieved when the total number of OH groups present was the same as glycerol. Additionally, when the structural isomers mannitol/sorbitol and ribitol/xylitol were compared at the same molarity (0.4 M), the isomer with the higher number of OH groups along the same side resulted in significantly higher levels of post-LN survival. We propose that the mode of action of polyalcohols is based not on molarity, but on the total number of OH groups present in the medium. Furthermore, based on these results we propose that the orientation of OH groups is a determining factor in effective cryopreservation.

Artificial Reduction of the Blastocoelic Cavity Increases the Survival of Blastocysts (BL) After Vitrification Protocol

Objective: The use of a sequential culture system enables human embryos to develop Bl stage efficiently. However, a relatively poor survival rate after cryopreservation of Bl or expanded Bl stage has been reported. As compared morphologically to early stage embryos, Bl and expanded Bl consist of a fluid-filled cavity, named the blastocoele, which may disturb cryopreservative potential due to ice crystal formation during the cooling step.

High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer.

Successful cryopreservation of mammalian oocytes would provide a steady source of materials for nuclear transfer and in vitro embryo production. Our goal was to develop an effective vitrification protocol to cryopreserve bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. Bovine oocytes matured in vitro were placed in 4% ethylene glycol (EG) in TCM 199 plus 20% fetal bovine serum (FBS) at 39 degrees C for 12-15 min, and then transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose in TCM 199 and 20% FBS). Oocytes were vitrified in microdrops on a precooled (-150 degrees C) metal surface (solid-surface vitrification). The vitrified microdrops were stored in liquid nitrogen and were either immediately thawed or were thawed after storage for 2-3 wk. Surviving oocytes were subjected to 1) parthenogenetic activation, 2) in vitro fertilization, or 3) nuclear transfer with cultured adult fibroblast cells. Treated oocytes were cultured in KSOM containing BSA or FBS for 9 to 10 days. Embryo development rates were recorded daily and morphologically high-quality blastocysts were cryopreserved for nuclear transfer-derived embryos at Day 7 or Day 8 of culture. Immediate survival of vitrified/thawed oocytes varied between 77% and 86%. Cleavage and blastocyst development rates of vitrified oocytes following in vitro fertilization or activation were lower than those of the controls. For nuclear transfer, however, vitrified oocytes supported embryonic development as equally well as fresh oocytes.

Overcoming a Permeability Barrier by Microinjecting Cryoprotectants into Zebrafish Embryos (Brachydanio rerio)

The goal of this research was to examine the developmental effects on zebrafish embryos (Brachydanio rerio) when cryoprotectants were directly microinjected into the yolk. Our objectives were to: (i) determine the final concentration of propylene glycol (PG) and dimethyl sulfoxide (Me2SO) that the embryos could tolerate without causing teratogenic effects; (ii) determine if the toxicity of Me2SO could be reduced by the simultaneous presence of various proportions of amides; and (iii) examine whether this intracellular cryoprotectant incorporation could reduce the cryodamage to the yolk syncytial layer (YSL) after vitrification trials. The rationale for conducting these microinjection experiments was to overcome the permeability barrier of the YSL. Intracellular PG produced better survival than Me2SO (P < 0.05). Embryos tolerated both 10- and 30-nl microinjections of PG, yielding final concentrations of 2.3 and 5.0 M within the yolk, resulting in 70 ± 3 and 35 ± 4% survival at day 5, respectively. In similar experiments with Me2SO, survival was lower than PG at 60 ± 4 and 14 ± 4% at 2.4 and 5.2 M. Unlike other cellular systems, the presence of amides, specifically acetamide or formamide, did not reduce the toxicity of Me2SO in zebrafish embryos (P > 0.05). During vitrification trials, we estimated a 25% dehydration of the yolk, yielding an effective PG concentration of 5.9 M. However, the incorporation of this vitrifiable concentration of PG was not sufficient to improve the postthaw morphology of the YSL (P > 0.05). Clearly, other factors need to be examined in establishing a successful vitrification protocol for zebrafish embryos.

Cryopreservation of Anigozanthos viridis ssp. viridis and related taxa from the south-west of Western Australia

Cryostorage of shoot apices of the perennial monocotyledonous species Anigozanthos viridis Endl. ssp. viridis (Haemodoraceae) was investigated by using a modified vitrification protocol. The highest post-thaw survival of shoot apices (41.4%) involved preculturing shoot apices on 0.4 M sorbitol for 48 h followed by incubation in a vitrification cryoprotective solution (PVS2) for 25 min at 0˚C. The level and type of cytokinin used in the culture stage was also found to influence cryostorage success with post-thaw survival decreasing from 41% with zero to low levels of cytokinins to below 5% for cytokinin levels that are typical of plant tissue applications (2.5 ˜M) for Australian plant species. Five Haemodoraceae taxa (Anigozanthos Labill. spp. and Conostylis R.Br. spp.) were successfully cryopreserved with this modified protocol; however, a sixth taxon, Macropidia fuliginosa (Hook.) Druce., remained unresponsive to this vitrification technique.

Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos

Embryo quality of in vitro-produced bovine blastocysts was assessed at several steps of a vitrification procedure in which glycerol and ethylene glycol were used as cryoprotectants (3-step equilibration with cryoprotectants followed by vitrification, dilution of the cryoprotectants in 0.85 M galactose then in embryo transfer freezing medium [ETF], and finally co-culture for periods). To visualize cell membrane alterations, double staining was performed using a cell permeant fluorochrome (bisbenzimide — BIS) and a nonpermeant one (propidium iodide — PI). In Experiment 1, the effect of the vitrification procedure on the hatching rate and total cell number was assessed 72 h after treatment. Hatching rate and the number of stained nuclei were decreased in comparison with untreated embryos when blastocysts were exposed to the whole procedure with or without vitrification (respectively 42 and 53% vs 76% for hatching and 128 ± 17 and 141 ± 17 vs 226 ± 13 for stained nuclei). In Experiment 2, the effect of cryoprotectants and their dilution was evaluated on membrane permeability and total cell numbers at various steps of the vitrification procedure. Blastocysts exposed only to cryoprotectant solutions and stained immediately after dilution in galactose showed no modification. After dilution in ETF, the total number of stained nuclei decreased, and the number of blastomeres showing membrane permeabilization (PI-stained) increased (respectively, 74 ± 5 vs 110 ± 5 and 32 ± 2% vs 0.1 ± 1.8%). In Experiment 3, we demonstrated that the total number of stained nuclei after ethanol fixation (membrane permeabilization) was higher when embryos treated up to dilution in ETF were stained with PI than when the same embryos were stained with BIS. This suggests that, for unknown reasons, some nuclei of the treated embryos were not stained with BIS. Membrane permeabilization and inability of BIS to stain some nuclei were the most obvious alterations probably induced by osmotic shock at dilution. This hypothesis is supported by the fact that the introduction of a further dilution step in 0.42M galactose (Experiment 4) before dilution in ETF decreased the proportion of cells permeant to PI and increased the hatching rate after 72 h of co-culture. In conclusion, double staining with BIS and PI allowed for discrimination between different types of cellular injuries after the various steps of our vitrification protocol. It represents a useful tool for adjusting equilibration and dilution conditions during a cryopreservation procedure.

Cryopreservation of Cattle Oocytes: Effects of Meiotic Stage, Cycloheximide Treatment, and Vitrification Procedure

Different parameters likely to influence the survival of bovine oocytes after a vitrification procedure were evaluated: oocyte meiotic stage, cycloheximide treatment at the beginning or the end of maturation, and three vitrification procedures using conventional straws, open pulled straws (OPS), or microdrops. For each procedure a mixture of cryoprotectants (25% ethylene glycol and 25% glycerol) was used. After the oocytes were warmed and subjected toin vitromaturation and fertilization, the number that developed into blastocysts was determined. Results show that cryoprotectant exposure reduced embryo development and that cycloheximide treatment had no beneficial effect on oocytes vitrified in conventional straws. Among the three vitrification procedures, only the OPS method yielded blastocysts (approximately 3% of vitrified oocytes) irrespective of their initial meiotic stage. This result highlights the major influence of the cooling rate in an oocyte vitrification protocol.

Cryopreservation of in vitro-grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification

Alginate coated meristems from in vitro-grown axillary buds of potato (Solanum tuberosum L.) were successfully cryopreserved by vitrification. Excised meristems were precultured on sucrose-enriched MS medium and then encapsulated. To induce dehydration tolerance (osmotolerance), encapsulated meristems were treated with a mixture of 2 M glycerol plus 0.6 M sucrose for 90 min. These encapsulated meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 hr at 0°C prior to a plunge into liquid nitrogen. Successfully vitrified meristems developed shoots within 3 weeks after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 70%. No difference was observed in RAPD analysis using 17 primers between cryopreserved and non-treated plantlets. The cryogenic protocol was successfully applied to 14 cultivars. It was also confirmed that the encapsulated vitrified meristems produced much greater shoot formation than the encapsulated dried meristems. Thus, the encapsulation vitrification protocol appears promising for cryopreservation of potato germplasm.

Cryopreservation of invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure.

Invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott.) were successfully cryopreserved by vitrification. Excised shoot tips precultured on solidified MS supplemented with 0.3M sucrose and maintained under a 16 h phtoperiod at 25°C for 16 h were loaded with a mixture of 2M glycerol plus 0.4M sucrose for 20 min at 25°C. The shoot tips were then sufficiently dehydrated with a highly concentrated vitrification solution (PVS2) for 20 min at 25°C prior to immersion into liquid nitrogen. Successfully vitrified and warmed shoot tips resumed growth within 7 days and developed shoots directly without intermediate callus formation. The average rate of shoot recovery amounted to around 80%, and the vitrification protocol appeared to be very promising for the cryopreservation of taro germplasm.

Cryopreservation of kunming mouse oocytes using slow cooling, ultrarapid cooling and vitrification protocols

The cryopreservation of oocytes has been only marginally successful with any of the current protocols, including slow cooling, rapid cooling and vitrification. We wished to test the hypothesis that oocytes from a single mouse strain would freeze successfully by 1 of the 3 mentioned protocols. Unfertilized Kunming mouse oocytes obtained 14 h after PMSG/hCG administration were randomly assigned to be cryopreserved after slow cooling, ultra rapid cooling and vitrification. Oocytes were thawed by straws being placed into 37 °C water, and their morphological appearance and in vitro fertilization capability were compared with that of oocytes that had not undergone cryopreservation. Survival of oocytes was indicated by the absence of darkened ooplasm or by broken membranes or zona pellucida. Functional integrity was evaluated by the formation of a 2-cell embryo after IVF. Survival rate of slow cooled oocytes did not differ from that seen in vitrified oocytes (55.1 vs 65.9%) but was significantly lower in the rapidly cooled oocytes (24.2%; P < 0.01). The results of IVF of slow cooled and vitrified oocytes were similar to those of the control group (72 and 73 vs 77%; P > 0.05). It appears that Kunming mouse oocytes can be successfully cryopreserved using the slow cooling method with 1,2-propanediol and vitrification, which contains both permeating and nonpermeating cryoprotectants.

Changes in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol.

A systematic approach was taken to assess and optimize a protocol for intracellular vitrification by introducing high concentrations of the cryoprotectant agent (CPA) ethylene glycol (EG) into unfertilized murine oocytes. The effects of EG on membrane integrity, microfilament organization, and developmental potential were evaluated. During exposure to 0.5-2 M EG, oocytes showed maximum shrinkage to 55.5% of the isotonic volume within the first minute and reexpanded to their initial volume within 15 min. Transferral of oocytes to higher concentrations of EG (4-8 M EG) for 1-5 min after 15 min of equilibration at 2 M EG was tolerated well. Microfilament organization appeared normal after this equilibration period. During prolonged exposure (> 5 min) to high concentrations of EG (> 4 M), membrane blebs were noticed on the surface of the cells, and microfilament distribution was disturbed. After treatment with 6 M EG and vitrification with 6 M EG + f2p40.5 M sucrose, there were no significant differences in development to the two-cell and blastocyst stages between CPA-treated, vitrified, and control oocytes. These results indicate that EG is an effective CPA for mouse oocyte vitrification protocols without any observed compromise in morphology and developmental functions.

Fertilization and early embryology: Synthetic and biological macromolecules: protection of mouse embryos during cryopreservation by vitrification

The ability of macromolecules to protect 8-cell mouse embryos during a vitrification protocol was assessed by the comparison of a synthetic macromolecule, polyvinylpyrrolidone (in the form of Percoll), and a biological macromolecule, human serum albumin (HSA). Vitrification solutions, which included glycerol (50% v/v), sucrose (0.75 mol/l) and either macromolecule Percoll (50% v/v) or HSA (1.125% w/v), were found to provide similar rates of survival. Both compounds resulted in a significant reduction in the incidence of disruption to the zonae pellucidae of cryopreserved embryos, to 4.8 +/- 1.3% and 10.3 +/- 1.2% respectively, when compared to the outcome when neither was present in the vitrification solution (20.4 +/- 2.5%; P < 0.01). Polyvinylpyrrolidone in the form of Percoll offers advantages over HSA in this work: it provides a lower rate of zona disruption and avoids the need for screening for pathogenic contaminants such as human immunodeficiency virus and hepatitis B and C.

Cryopreservation of kunming mouse oocytes using slow cooling, ultrarapid cooling, and vitrification protocols

Cryopreservation of unfertilized mouse ova and 2-cell embryos by a vitrification technique was examined. Survival was defined by development to the hatching blastocyst stage after in vitro fertilization. With 19 embryos at the 2-cell stage, the authors obtained 100% morphologic survival and 89% development to hatching blastocyst stage. To define the optimal conditions for vitrification of ova, the authors treated a total of 845 unfertilized ova. In experiments done at 0°C, the concentration of vitrification solution (VS1) and the length of exposure of ova to VS1 both had significant (P < 0.01) effects on survival. The mean survival rate for controls in ten experiments was 52%. VS1 100% or 90% in HEPES buffered saline and 10 minutes’ exposure yielded rates that did not differ significantly from controls. Significantly lower survival rates followed the use of 70 and 80% solution and exposure for 5, 15, 20, or 30 minutes. Thus, under these conditions, exposure of unfertilized mouse ova to VSl and cooling to 0°C did not interfere with in vitro fertilization and development of embryos. However, in five experiments in which a total of 101 ova were plunged into liquid nitrogen after treatment with VS1 under the optimal conditions, none could be fertilized in vitro.

Cryopreservation of equine oocytes by 2-step freezing

Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz micro-capillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1–2 M). The cryoprotectants used included 2 M 1,2-propanediol (PROH, cell membrane permeable) and 0.5 M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post-vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2 M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells.

The preregistration year.

<h3>Abstract</h3> Photosynthetic dinoflagellates that live in symbiosis with corals (family Symbiodiniaceae) are fundamental for the survival of coral reef ecosystems. During coral bleaching events, it is assumed that these symbionts remain available in the water column, in sediments, or are seeded from unbleached coral colonies. Yet, this hypothesis has not been verified and it remains unclear whether some diversity of Symbiodiniaceae may be lost in the process. Culture methods have been developed for some Symbiodiniaceae, but for the vast majority of these photosynthetic symbionts, known culture methods are not successful at maintaining them for extensive periods. For these unculturable symbionts, cryopreservation, which places cells and tissues in suspended animation for days to decades, offers the best hope for saving the biodiversity of these crucial coral partners. Some cryopreservation processes use slow freezing, but if the cells are sensitive to low temperatures, as is the case for Symbiodiniaceae, then rapid freezing, called vitrification, is needed. We here, tested two published vitrification protocols that had been designed for algal symbionts extracted from Hawaiian corals, but we were unable to recover living symbionts after vitrification and warming. Therefore, we report a successful optimisation of the former vitrification protocols, which we tested on algal symbionts freshly extracted from three Hawaiian coral species, the development of ultra-rapid laser-warming cryopreservation techniques for symbionts, and banking procedures for algal symbionts. We also present some successful uptake of cryopreserved algal symbionts by coral larvae, although at a low rate. It is unclear why the former vitrification protocols failed but we propose that it may have been related to thermal stress and bleaching events that occurred on several occasions throughout the Hawaiian Islands. Maintenance of biodiversity is essential for sustaining functional, productive ecosystems with the adaptability to effectively recover from disturbances. By successfully cryopreserving and banking coral symbionts, we provide a critically needed component for securing Symbiodiniaceae biodiversity into the future.

Co-culture conditions and partial zona dissection affect hatching rates of thawed bovine blastocysts produced In vitro: A preliminary report

Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experiments. In experiment 1, the effect of either one- or two-puncture treatments before aspiration of blastocoel fluid and exposure to vitrification solutions was evaluated. No difference was detected in mean embryo volume across treatment groups after exposure to vitrification solutions or after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increase at 48 and 72 hours during in vitro culture were 100%, 83%, and 75% compared with 93%, 67%, and 50% for one- and two-puncture treatment groups, respectively. Capsule loss was 25% for one-puncture and 50% for two-puncture treatment groups. In experiment 2, no difference was detected in mean embryo volume for indirect introduction (aspiration of blastocoel fluid + equilibration) and direct introduction (injection of cryoprotectant into blastocoel cavity) treatment groups, after exposure to dilution solution or to culture medium. There was no difference in mean embryo volume for the indirect and direct introduction treatment groups after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increases at 48 and 72 hours during in vitro culture were 100%, 76.9%, and 69.2%, respectively, for both treatment groups. Those embryos subjected to the direct introduction treatment had a higher (P = 0.05) percent capsule loss (70%) compared with the indirect introduction treatment group (31%). The pregnancy rate after transfer of vitrified expanded Grade 1 blastocysts using the indirect introduction method was 83% (5/6). Three pregnancies were allowed to continue to term and resulted in the birth of three healthy foals. The vitrification protocol used in this study has the potential to become a key tool for the successful cryopreservation of equine expanded blastocysts.

Vitrification of mouse oocytes using short cryoprotectant exposure: effects of varying exposure times on survival.

The effects on oocyte viability of varying the duration of exposure to cryoprotectants before rapid cooling to -196 degrees C were examined, using the vitrification protocol of Nakagata. A very short exposure (15 sec) was found to be optimal, resulting in an overall rate of development from vitrified oocytes to hatching blastocysts of 31.8%. Very high rates of survival (77-89%) of oocytes exposed to the cryoprotectant media, but without the vitrification, together with extreme variability in results between straws in the vitrified groups, suggest that losses in viability during vitrification may result from ice damage during devitrification of the medium.