Cryologic Vitrification Method Research Articles

“Comparison of two closed vitrification methods for vitrifying dromedary camel (Camelus dromedarius) embryos"

A total of 184 dromedary camel embryos were vitrified using a novel vitrification kit specifically developed for camel embryos. These embryos were vitrified using a 3-step process by exposing them to vitrification solutions (VS) containing 20% foetal calf serum (FCS) with (+) or without (−) the addition of bovine serum albumin (BSA). Embryos were then further divided into two groups (<or ≥ 500 μm), vitrified using a closed vitrification system and stored on a Cryolock® embryo storage device or vitrified using solid surface vitrification of the Cryologic Vitrification Method (CVM)™ and stored on a Fibreplug™ embryo storage device. Embryos were then warmed and transferred in pairs, of approximately equal size, into recipient camels on Day 6 after ovulation. A total of 92 embryos were vitrified using the Cryolock; 86 embryos (C-BSA, n = 46; C + BSA, n = 40) survived warming, and were then transferred into 43 recipients. The pregnancy rate (PR) per recipient was 17% (C-BSA: 4/23) and 45% (C + BSA: 9/20) at 20 days after transfer but reduced to 9% and 30% at 60 days of gestation, respectively. For the Fibreplug group, a total of 92 embryos were vitrified, 86 embryos (F-BSA, n = 44; F + BSA, n = 42) survived warming, and were transferred into 43 recipients. The PR per recipient was 73% (F-BSA: 16/22) and 48% (F + BSA: 10/21) at 20 days after transfer but reduced to 50% (11/22) and 33% (7/21) at 60 days of gestation, respectively. Of the 39 pregnancies at 20 days after transfer, 77% (30/39) resulted from embryos with a diameter of 300–450 μm and 23% (9/39) were from embryos with a diameter of 500–850 μm. In conclusion, 1) significantly higher pregnancy rates were achieved when embryos were vitrified using the Fibreplug than using the Cryolock; 2) the effect of BSA on embryo vitrification is dependent on embryo storage device type and embryo diameter; 3) smaller vitrified embryos (<500 μm) tend to produce higher pregnancy rates than larger embryos (≥500 μm), however, the negative effect of embryo size on embryo vitrification is reduced when embryos are frozen using the original novel vitrification kit (no BSA) solid surface vitrification using the CVM.

Cryopreservation of human induced pluripotent stem cells by using a new CryoLogic vitrification method

Human induced pluripotent stem cells (hiPSCs) have the properties of differentiation potential and unlimited self-renewal. Developing efficient and highly safe methods to preserve hiPSCs is important due to they have demonstrated tremendous promise in disease etiology, drug discovery, and regenerative medicine applications. Traditionally, open systems for cell cryopreservation, such as conventional slow freezing and vitrification methods, were widespread application in the storage and transportation of hiPSCs. However, these two methods have such problems of low recovery rate and the risk of cross-contamination. Recently, closed systems for cell cryopreservation, such as CryoLogic Vitrification Method (CVM), were introduced to store and transport embryos. In this study, we developed a new friendly CVM by loading a small piece of hiPSCs colonies in the vitrification solution to the hook of Fiberplug to increase the cooling rate. To warm them, the CVM Fiberplug was immersed directly in a 37 °C warming solution for 1 min, and hiPSCs were then transferred to mTeSR1 medium. The result revealed that the new CVM had a high recovery rate and maintained the stemness and differentiation potential of hiPSCs. Our new CVM not only provide a safe way for hiPSCs preservation but also has a high survival rate in the storage of hiPSCs.

93 BLASTOCYST BISECTION TO MULTIPLY BIOPSIED AND VITRIFIED BOVINE EMBRYOS

Dairy cattle breeding schemes increasingly integrate embryo-based genomic selection to accelerate genetic gain. In contrast to the single offspring produced with conventional animal-based genomic selection, multifactorial IVF between elite parents increases genotypes for selection. Genetically superior embryos are identified from biopsies, and only those with the desired genotypes are transferred. To manage the logistics of such schemes, and enable seasonally born progeny, the cryo-preservation of embryos after biopsy and before embryo transfer is critical. Here, we compare 2 methods of cryo-preserving biopsied Day 7 blastocysts and report results from bisecting blastocysts to increase the number of selected embryos for transfer. Abattoir-sourced oocytes were matured in vitro and fertilized with sperm from a single sire. Embryos were cultured for 7 days in a modified Synthetic Oviduct Fluid medium. Approximately 15 cells were biopsied from the mural trophectoderm of grade 1 and 2 blastocysts in Embryo Hold medium minus BSA, using a micro-surgical blade (Bioniche Animal Health, Athens, GA, USA). Following biopsy, each blastocyst was cultured in Embryo Hold with 3 mg mL−1 BSA for ~2 h at 38.5°C to allow for re-expansion. In Experiment 1, embryos were randomly assigned to 1 of 2 cryo-preservation treatments: conventional slow freezing or the Cryologic vitrification method (CVM). Slow freezing entailed freezing in 1.5 M ethylene glycol and 0.1 M sucrose. The CVM involved a 2-step vitrification protocol, with 15% of both ethylene glycol and dimethyl sulphoxide in the final solution comprising Embryo Hold, 20% FCS, 1 M sucrose, and 0.1 mM Ficoll (GE Healthcare). Selected embryos were thawed/warmed and transferred in pairs to the uterine horn ipsilateral to the corpus luteum of each synchronized recipient heifer. In Experiment 2, each biopsied blastocyst was individually vitrified using CVM. Following warming, blastocysts were bisected into approximately equal halves. After ~2 h recovery, pairs of demi-embryos were transferred to recipients categorized with either normal (&gt;2.5, &lt;7 ng mL−1) or low (≥2, &lt;2.5 ng mL−1) plasma progesterone concentrations on Day 5 after oestrus. Embryo survival in both experiments was monitored by ultrasonography of fetal heartbeats up to Day 65 of gestation. Statistical significance was determined using Fisher’s exact test. In Experiment 1, embryo survival on Day 65 was significantly greater with CVM than slow freezing (25/54 = 46% v. 9/54 = 17%; P = 0.002). In Experiment 2, there was no significant difference in the number of fetuses as a percentage of original blastocysts, regardless of normal versus low progesterone levels (13/22 = 59% v. 4/9 = 44%, respectively). In conclusion, vitrification is superior for cryo-preserving biopsied blastocysts, possibly reducing cryo-damage compared with conventional slow freezing, and achieves rates of in vivo development similar to fresh IVF embryos. Embryo bisection potentially provides only a modest increase in the probability of generating a calf from each valuable, genomically selected embryo. Improving embryo competency and other methods of multiplication may maximize this likelihood.

43 NEW METHOD FOR ONE-STEP WARMING/IN-STRAW CRYOPROTECTANT DILUTION FOR IN VITRO-PRODUCED BOVINE BLASTOCYSTS AFTER VITRIFICATION WITH THE CRYOLOGIC VITRIFICATION METHOD

Vitrification is considered an alternative to slow-rate freezing to cryopreserve in vitro-produced (IVP) bovine embryos. However, the use of vitrified IVP embryos for embryo transfer under field conditions is difficult because of the requirements of the current thawing protocols. The objective of this study was to develop a simple one-step warming/in-straw cryoprotectant dilution procedure for IVP bovine blastocysts that were vitrified using the cryologic vitrification method. In this study, 109 Day-7 IVP blastocysts were subjected to vitrification using the conventional fibreplugs (groups of 5 embryos were loaded in 3 mL of vitrification medium). Warming was performed in one-step in MS1 (0.25 M sucrose in BV = TCM 199-Hepes + 20% FCS) either using a 4-well plate for 5 min (control group) or in a new system that allowed in-straw cryoprotectant dilution designed to avoid losses of embryos and to maintain the temperature required during this procedure. This new system is composed of an adaptor with a wider opening that is coupled to the French straw and a heated metal chamber to protect and keep the straw at 41°C. Warmed embryos were washed and subsequently cultured in mSOFaaci + 6 gL–1 BSA + 10% FCS for 48 h. Re-expansion (at 2, 24, and 48 h) and hatching rates (at 24 and 48 h) were recorded. Data were analysed by ANOVA and are presented as LSM ± standard error. Embryo survival rates of embryos warmed by the one-step warming/in-straw cryoprotectant dilution procedure did not differ from the control group (see Table 1). These results suggest that the cryologic vitrification method combined with our warming system for in-straw cryoprotectant dilution may be used for direct embryo transfer under field conditions. Table 1.Embryo survival rates of in vitro-produced embryos vitrified by the cryologic vitrification method and warmed by the new one-step warming/in-straw cryoprotectant dilution procedure This study received grant support: INIA-RTA 2011–0090 and FEDER. M. Muñoz was supported by grant MICINN-RYC08-03454, and B. Trigal by a grant from Cajastur. The authors are members of the COST Action FA1201 Epiconcept.

54 CRYOPRESERVATION OF BOVINE IN VITRO-PRODUCED EMBRYOS: INTRINSIC FACTORS DETERMINING VITRIFICATION OUTCOMES

As a part of a study on bovine embryo genomic evaluation, effects of an intact (iZP) or opened zonae pellucidae (oZP) of in vitro-produced embryos on vitrification outcomes were assessed. In the first experiment, only iZP embryos were subjected to vitrification, using either the hollow fibre vitrification method (HFV; Matsunari et al. 2012 J. Reprod. Dev.) or the cryologic vitrification method (CVM; CryoLogic®, Blackburn, Victoria, Australia). Developmental stage (morula = M; early blastocyst = EaB; blastocyst = B) and quality (good = 1; fair = 2) before vitrification were evaluated. In a first set of experiments, quality 1 and 2 iZP embryos were vitrified either by the HFV or the CVM method. A significant difference between the 2 methods was found when comparing overall survival rates (24–48 h post-thaw; HFV 59.32% v. CVM 78.9%; P &lt; 0.001; unpaired t-test, GraphPad Prism, GraphPad Software Inc., La Jolla, CA, USA). Concerning hatching rates, no significant difference was found (HFV 48.7% v. CVM 57.8%; P &gt; 0.1). In order to evaluate influence of embryo stage and quality on HFV or CVM outcomes, iZP embryos vitrified by the HFV method were classified regarding quality (1: n = 207; 2: n = 66; total n = 273) and embryo stage (M: n = 78; EaB: n = 74; B: n = 121). Concerning survival rates, no significant difference was found between M 1 (57.4%) and M 2 (44.6%), or EaB 1 (80%) and EaB 2 (68%). However, a significant difference was found when comparing B 1 (77%) and B 2 (29%; P &lt; 0.05). With regard to hatching rate, no significant difference was found between M 1 (39.9%) and M 2 (30%), or EaB 1 (66.6%) and EaB 2 (57%). However, significant difference was found between B 1 (60.9%) and B 2 (22%; P &lt; 0.05). With regard to the CVM (1: n = 172; 2: n = 82; total n = 254), no significant difference was found when analysing survival rates of EaB 1 (82%) and EaB 2 (80%), or B 1 (87%) and B 2 (74%), whereas the survival rates were significantly different between M 1 (80%) and M 2 (53%; P &lt; 0.05). Significant differences regarding hatching rates were not found between M 1 (51%) and M 2 (28%) or EaB 1 (55%) and EaB 2 (65%), whereas the hatching rate of B 1 (76%) was not significantly higher than that of B 2 (48.8%; P &lt; 0.05). In a second set of experiments, oZP EaB (1: n = 14) and oZP B (1: n = 30) were vitrified by the CVM method. No significant difference regarding survival rates concerning stage (95 and 72%, EaB and B, respectively), or between treatments (EaB iZP v. oZP, and B iZP v. oZP; P &gt; 0.1) was found. Effect of ZP, either intact or opened, does not seem to affect survival rates (judged by their re-expansion 24–48 h post-thaw) of good-quality embryos.The authors thank the Bavarian Research Foundation for the financial support (AZ-1031-1; DOK-153-12).

Survival of vitrified in vitro–produced bovine embryos after a one-step warming in-straw cryoprotectant dilution procedure

Vitrification is an alternative to slow-rate freezing for cryopreserving bovine embryos. However, this technology requires simplification if it is to be used under field conditions. The main objective of this work was to develop a new system for the direct transfer of vitrified embryos to be used under farm conditions. For this, three objectives were set: (1) to compare the effect of vitrification, using the cryologic vitrification method (CVM), and slow-rate freezing on bovine embryo development and quality; (2) to develop a one-step warming procedure for bovine in vitro–produced (IVP) vitrified (by CVM) embryos; and (3) to assess the effects on embryo survival of a new method for the direct transfer of vitrified IVP bovine blastocysts. In vitro–produced blastocysts were initially either vitrified by CVM or subjected to slow freezing to compare embryo survival and quality (experiment 1). No differences were detected between these cryopreservation techniques in terms of the survival and quality variables at 24 hours or in terms of the proteins expressed. However, at 48 hours the vitrified embryos showed higher hatching rates, greater total cell numbers, and lower apoptotic indices (P < 0.05). In experiment 2, CVM-vitrified IVP blastocysts were warmed by the conventional two-step or one-step warming procedure by incubating them at 41 °C in 0.25 M sucrose for 10 minutes, 0.15 M sucrose for 10 minutes, or 0.25 M sucrose for 5 minutes. In addition, embryo transfer (ET) was performed using vitrified embryos warmed by the one-step procedure in 0.25 M sucrose solution for 5 minutes. As a control group, IVP fresh embryos were transferred to recipient females. No differences were observed in embryo survival or total cell number between any of the warming procedures. Moreover, no significant differences for pregnancy at 60 days were found between the ET groups. In experiment 3, expanded IVP blastocysts were then either vitrified using a conventional or a modified fiber plug designed to allow direct ET after in-straw cryoprotectant (CP) dilution. They were warmed using the one-step process (0.25 M sucrose, 5 minutes) in a 0.25 mL French straw. Embryo recovery associated with the modified fibreplug system was less reliable than with the conventional system. However, no differences were seen between the systems in terms of in vitro embryo survival among those finally recovered. Finally, IVP blastocysts were vitrified using conventional fibreplugs to maintain a high embryo recovery rate, and then warmed using the one-step warming in-straw CP dilution procedure, but using an adapter with a wider opening coupled to the French straw and a heated metal chamber to protect and keep the straw at 41 °C (experiment 4). No differences were seen in embryo survival rates between the two groups. The CVM combined with this new one-step warming in-straw CP dilution procedure could be used for direct ET under field conditions.

Closed vitrification of mouse oocytes using the CryoLogic vitrification method: A modification that improves developmental competence

ObjectiveTo compare the mouse oocyte vitrification outcomes of the CryoLogic vitrification method (CVM) and the conventional open method using a Cryotop. Two CVM methods (original CVM and modified CVM) were tested.MethodsMature oocytes obtained from female BDF-1 mice were vitrified by two-step exposure to equilibrium and vitrification solutions. Three vitrification protocols were tested on three groups: the CVM-kit, modified CVM, and Cryotop groups. After exposure to the two solutions, the oocytes were vitrified. After warming, the oocytes were fertilized in vitro, and the embryo development was assessed. Blastomeres positive for caspase were counted using an in situ assay kit. The spindle morphology and chromosome configurations of warmed vitrified oocytes were also assessed.ResultsThe modified CVM and Cryotop groups showed similar developmental capacities, and similar proportions of cells with intact spindles and chromosome configurations. The modified CVM protocol was superior to the original CVM protocol for developmental competence and intact spindle preservation. However, the CVM group showed a relatively higher number of apoptotic cells in blastocysts.ConclusionClosed vitrification using the modified CVM protocol may be used as an alternative to the conventional open method, but strategies to decrease apoptosis in the blastomere need to be investigated.

63 QUALITY OF BOVINE EMBRYOS PRODUCED IN VITRO FROM IMMATURE OOCYTES TREATED WITH A SUBLETHAL HYDROSTATIC PRESSURE

High hydrostatic pressure (HHP) treatment of immature porcine oocytes improves embryo development rates and cell numbers (Pribenszky et al. 2008 Anim. Reprod. Sci. 106, 200–207). However, it is unknown if similar effects can be obtained with bovine oocytes and how HHP affects cryopreservation of the developed blastocysts. In this work, we analyzed the effect of an HHP treatment (Cryo-Innovation Ltd., Budapest, Hungary) on bovine cumulus–oocyte complex (COC) as determined by their developmental ability and embryo quality. Immature COC were submitted to a pressure treatment (200 bar, 1 h at 37°C; HHP group; n = 643) in HEPES-buffered TCM199. Simultaneously, a group of COC was held at 37°C for 1 h (T group; n = 304) in HEPES-buffered TCM199, while other COC were untreated (n = 1182). After in vitro maturation, COC were fertilized in vitro (IVF) and cultured in modified SOF + 6 g L–1 BSA (Holm et al. 1999 Theriogenology 52, 683–700), and embryo development was recorded (5 replicates). Day 7 and 8 excellent- and good-quality embryos were selected for vitrification (cryologic vitrification method; Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). After warming, vitrified blastocysts were cultured in modified SOF + 6 g L–1 BSA + 10% FCS for 48 h (3 replicates). Those blastocysts hatching after warming (at 24 and 48 h) were fixed and stained for differential cell counts. Data were analyzed by ANOVA and REGWQ test and are presented as least squares means ± standard error. The HHP-treated oocytes showed increased development rates on Day 3 (Day 3 ≥5-cell embryos: 64.5 ± 2.9a, 53.4 ± 3.9b, 56.7 ± 2.2b for HHP, T, and untreated groups, respectively; a v. b: P &lt; 0.05); however, D8 blastocyst rates were not affected by the pressure treatment (28.5 ± 1.6, 26.4 ± 2.2, and 27.8 ± 1.3 for HHP, T, and untreated groups, respectively). Treatment did not affect survival rates to vitrification (2-h re-expansion rates: 100 ± 6.7, 100 ± 6.7, and 95.4 ± 6.7; 48-h hatching rates: 58.1 ± 9.4, 71.2 ± 9.4, and 62.3 ± 9.4, for HHP, T, and untreated, respectively). Embryos that hatched after warming did not differ in inner cell mass and trophectoderm cell counts (inner cell mass: 15.0 ± 1.9, 12.7 ± 3.0, and 13.0 ± 2.0; trophectoderm: 133.6 ± 8.4, 137.3 ± 12.8, and 138.4 ± 8.6 for HHP, T, and untreated groups, respectively; P &gt; 0.05). Complementary studies are needed to analyze the effects of a sublethal stress in bovine oocytes on the subsequent embryo production and quality. Species-specific mechanisms could underlie the differences in results obtained in bovine and porcine. RTA2011-00090 (FEDER-INIA). Muñoz, Trigal, and Correia are sponsored by RYC08-03454, Cajastur, and FPU2009-5265, respectively.

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirmed an individual effect of the bull as well as development rates of embryos produced with sorted sperm lower than embryos with unsorted sperm, independent of the culture system used. The cryoresistance to vitrification of embryos produced with sexed sperm did not differ from that of conventionally produced embryos (re-expansion rates at 24 and 48 h: 74.6% vs. 75.5%, and 64.5% vs. 68.1% for embryos produced with conventional and sorted sperm, respectively; hatching rates at 48 h: 63.55% vs. 55.5% for embryos produced with conventional and sorted sperm, respectively). Finally, no significant differences were found in pregnancy rates after the embryo transfer of fresh and vitrified/warmed blastocysts (52.8% vs. 42.0%, respectively; P > 0.05). Male and female embryos produced with sorted sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. Our culture system, coupled with the vitrification in fiber plugs, provides good quality sex-known embryos which survive vitrification at similar rates than embryos produced with conventional unsorted sperm; also it produces good pregnancy rates after transfer of sexed embryos both fresh and after vitrification and warming.

Cell Counts and Survival to Vitrification of Bovine In Vitro Produced Blastocysts Subjected to Sublethal High Hydrostatic Pressure

This work analyses the effects of a high hydrostatic pressure (HHP) treatment on in vitro survival of in vitro produced (IVP) bovine embryos vitrified with the Cryologic Vitrification Method (CVM). Consequences on embryo quality in terms of cell proliferation and differentiation, and levels of embryonic Heat Shock Protein 70 (Hsp-70) were also examined. Day 7 and 8 bovine in vitro-produced blastocysts were submitted to an HHP treatment (60 MPa, at 32 °C for 1 h) and allowed to recover for 1 or 2 h in culture medium. The HHP treatment did not improve blastocyst survival rates after vitrification/warming. Survival (24 h post-warming) and hatching (48 h post-warming) rates were 79.3 ± 4.9 and 51.8 ± 4.2 vs 73.9 ± 4.2 and 44.7 ± 4.1 for untreated controls and HHP-treated embryos, respectively. Total cell numbers measured in fresh embryos were reduced after 1 h at 32 °C, with or without HHP treatment, indicating that cell proliferation was stopped as a result of stress. Vitrified HHP-treated embryos that hatched at 48 h after warming showed increased cell numbers in their ICM compared with untreated controls (50.2 ± 3.1 vs 38.8 ± 2.7), indicating higher embryo quality. Treatment of blastocysts with HHP did not alter the level of the Hsp-70 protein. In our conditions, HHP treatment did not affect the cryoresistance of these embryos. However, combination of HHP treatment and vitrification in fibreplugs resulted in an increase in the ICM cell number of hatched embryos 48 h post-warming.

Piglets produced from in vivo blastocysts vitrified using the Cryologic Vitrification Method (solid surface vitrification) and a sealed storage container

The objective was to develop a simple successful porcine cryopreservation protocol that prevented contact between embryos and liquid nitrogen, avoiding potential contamination risks. In vivo-derived blastocysts were collected surgically from donor pigs, and two porcine embryo vitrification protocols (one used centrifugation to polarize intracytoplasmic lipids, whereas the other did not) were compared using the Cryologic Vitrification Method (CVM), which used solid surface vitrification. The CVM allowed embryos to be vitrified, without any contact between embryos and liquid nitrogen. Both protocols resulted in similar in vitro survival rates (90% and 94%) and cell number (89 ± 5 and 99 ± 5) after 48 h in vitro culture of vitrified and warmed blastocysts. The protocol that did not use centrifugation was selected for continued use. To protect vitrified embryos from contact with liquid nitrogen and potential contamination during storage, a sealed outer container was developed. Use of this sealed outer container did not affect in vitro survival of cryopreserved blastocysts. In vivo blastocysts (n = 151) were collected, vitrified, and stored using the selected protocol and sealed container. These embryos were subsequently warmed and transferred to six recipients; five became pregnant and farrowed a total of 26 piglets. This embryo vitrification method allowed porcine embryos to be successfully vitrified and stored without any contact with liquid nitrogen.

Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing

This study was designed to asses murine preantral follicle survival and growth, after cryopreservation of ovarian tissue by two different methodologies, solid-surface vitrification by the Cryologic vitrification method (CVM) and slow-rate freezing (SRF). Cryotreated tissue was stored in liquid nitrogen for 24 h, and upon warming follicle viability was assessed by live/dead fluorescent probes, and by 7-day autotransplantation of both cryotreated tissue types to the left and right kidney capsule of the donor animals ( n = 16). The live/dead assay immediately upon tissue warming did not allow a distinction to be made in terms of follicle viability between the CVM and SRF cryoprocedure. In grafted tissue, follicular survival and growth was assessed by conventional histological examination and proliferating cell nuclear antigen immunohistochemistry. In each experimental group (control, CVM and SRF), follicles were classified according to developmental stage, and a comparison of the proportions of follicle stages between the three groups was executed by statistical analysis of variance. The fraction of primordial follicles in CVM and SRF grafts significantly decreased as compared to control tissue, whereas intermediary and primary follicles significantly increased. The proportion of secondary and antral follicles after SRF was significantly larger than after CVM, but did not differ significantly between CVM and control tissue. The observed massive follicle activation is a typical transplantation effect, but testifies to the survival of cryopreserved follicles. In both types of cryotreated tissue, growing follicles, including antral stage, were present in grafts from all recipient animals. The significantly more abundant further developed stages in SRF treated tissue, however, suggest that CVM treated tissue may have suffered a growth disadvantage. To our knowledge, this is the first time that the CVM technique has been utilized to vitrify preantral follicles.

66 INDIRECT SOLID-SURFACE VITRIFICATION OF MURINE OVARIAN BIOPSIES AND ISOLATED FOLLICLES

Vitrification is a quick and relatively easy cryopreservation technique, but requires high cryoprotectant concentrations and extremely high cooling rates. Possible disadvantages include contamination of specimens with liquid nitrogen (LN2)-born pathogens and compromised post-thaw survival due to cryoprotectant toxicity and/or osmotic shock. The aims of the current research were to vitrify murine ovarian biopsies and isolated follicles using the Cryologic Vitrification Method 1 (CVM1™, Linde Gas Cryoservices, Hedel, The Netherlands), and assess tissue viability after thawing. Ovaries from 5 mice were sectioned into small biopsies (≤0.5 mm3), and ovaries from 5 additional animals were enzymatically digested (collagenase 1 mg mL–1) and filtered over a 100-µm sieve to isolate preantral follicles. Specimens were exposed to a series of vitrification solutions at 25�C, with the final solution consisting of sucrose (0.6 m), ethylene glycol (20%), and DMSO (20%). After 20 to 40 s of equilibration in the final medium, specimens were loaded into 3-µL droplets onto the hook of a plastic Fibreplug™. Droplets were vitrified by contact with the surface of the CVM1 metal block, and pre-cooled by partial submersion in LN2. Fibreplugs were subsequently inserted into pre-chilled straws and plunged into LN2 for 24 h. Specimens were warmed by removing the Fibreplug from the straw; the vitrified droplets were immersed in a series of sucrose solutions (0.3 m, 0.3 m, and 0.2 m) for 5 min at 25�C, and then transferred to PBS supplemented with 5 mg mL–1 BSA. Post-warming viability of isolated follicles and biopsies was assessed by the fluorescent live/dead probe calcein-ethidium. Follicle survival was also appraised in vivo by autotransplantation of either devitrified isolated follicles or biopsies (6 to 12) to the left kidney capsule of the donor animals. The recipients were euthanized 7 days posttransplantation by CO2 inhalation. Grafted tissue was fixed, paraffin-embedded, serially sectioned, and stained with hematoxylin-eosin for histological evaluation. Additional slides were immunohistochemically stained with proliferating cell nuclear antigen (PCNA) to differentiate between growing and non-growing follicles. In all digested samples, more than 80% of thawed isolated follicles were viable, as demonstrated by live/dead staining. In addition, fluorescent probes confirmed the presence of live follicles in 100% of thawed biopsies. In each of the 5 biopsy-transplanted animals, several biopsies survived, and in 3 mice grafted with isolated follicles, vital tissue could be retrieved. Histological examination of the 8 grafts revealed PCNA-positive follicles. These results indicate that indirect solid-surface vitrification of murine ovarian biopsies and isolated preantral follicles is a viable option to slow-rate freezing. The growing follicles in transplanted frozen–thawed tissue confirm the viability of surviving follicles. As specimens do not come into direct contact with LN2 during the vitrification process, the contamination risk is greatly reduced.

P-206: Non-immersion vitrification - a viable alternative to slow freezing of blastocysts

Slow freezing of human blastocysts is a widely used technique in ART but is often associated with a high rate of cell lysis leading to decreased embryo viability. It is time consuming and also has high equipment costs. In contrast, vitrification reduces the risk of ice crystal formation hence reducing cell lysis, has shown promising implantation rates in many species including human, is rapid and relatively inexpensive. In this ongoing study we compare the preliminary clinical results using the Cryologic Vitrification Method 1 (CVM 1) with historical data using a standard slow freezing procedure. Oocytes were fertilised and cultured in the Sydney IVF suite of sequential media (Cook IVF, Brisbane) for up to 144 hours. Day 5/6 supernumerary blastocysts produced prior to January 2006 were frozen using a programmed control rate freezer (Planer, UK) in Sydney IVF blastocyst freezing media. Slow freeze embryos were passaged through a series of ambient temperature solutions containing glycerol (9%) and sucrose (0.2M) in the final solution, transferred into embryo freeze straws, placed into a programmed freezer and then seeded manually. From January 2006 all blastocysts were vitrified In Sydney IVF Vitrification Media using the CVM 1 kit (Cryologic, Australia). Embryos were passaged through a series of vitrification solutions at 37°C, with the final solution containing trehalose (0.68 M), ethylene glycol (16%) and DMSO (16%) . Within 30 to 40 seconds in this final vitrification solution blastocysts were loaded in a 2-3 μl drop onto a fine plastic hook at the end of a fibreplug. This microdrop was vitrified by placing it onto the surface of the liquid nitrogen pre-cooled metal vitrification block and were then stored in sealable plastic straws. The blastocysts did not have direct contact with liquid nitrogen at any stage. Straws containing the slow freeze embryos were thawed in air for 30 seconds then in a water bath (30°C) for 30 seconds and then were transferred into a series of thawing solutions containing varying concentrations of sucrose at ambient temperature. Vitrified blastocysts were thawed by plunging the hook containing the vitrified embryo directly into the warming solution at 37°C. Historically, most pregnancies (55%) obtained using our slow freezing protocol were from embryos that had 90 -100% cell survival. Our 2005 data indicates that 41% (432/1047) of slow frozen embryos were in this survival band. Preliminary 2006 data indicates that a higher embryo survival rate of 79% (19/24) of vitrified embryos had cell survival of 90% or greater, (p < 0.005). To date we have obtained 10 +βhCG pregnancies from 18 embryo transfers (56%, 1.1 embryos per transfer). Comparative data from slow frozen embryos in 2005 gave a +βhCG pregnancy rate of 34% (222/658), transferring on average1.3 embryos, (p=0.095). These results indicate that non-immersion vitrification of human blastocysts could provide a safe and viable alternative to current slow freezing protocols. High post-thaw survival and pregnancy rates are necessary to encourage an increase in the proportion of cases where single embryo transfer (SET) is the preferred protocol to avoid multiple pregnancies [Fertil. Steril. 2005;84: 1620].

97 ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS FOLLOWING VITRIFICATION BY CVM OR SLOW FREEZING WITH ETHYLENE GLYCOL AND TRIPLE TRANSFER

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P &lt; 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P &lt; 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).

243 PREGNANCY RATES IN THE FIELD AFTER THE TRANSFER OF BOVINE IVP EMBRYOS VITRIFIED BY THE CRYOLOGIC VITRIFICATION METHOD

Although large numbers of IVP embryos can be produced from donor cattle in a short period of time, commercial acceptance of the technology depends on the ability to cryopreserve these embryos and achieve a 50% pregnancy rate in large-scale embryo transfer programs. Many studies have reported low pregnancy rates of about 20% after the transfer of cryopreserved IVP embryos. We have developed the user friendly CryoLogic Vitrification Method (CVM) that vitrifies embryos on a solid surface at −196°C and warms them rapidly in a one-step procedure prior to transfer (Lindemans et al. 2004 Reprod. Fertil. Dev. 16, 174). We present an overview of the pregnancy rates in the field after vitrification of bovine IVP embryos by the CVM. The bos taurus IVP embryos in southern Australia (bT) and the bos indicus-based IVP embryos in northern Australia (bI) were produced by our standard TVR and IVP methodology (Fry et al., 2003 Theriogenology 59, 446). Pregnancy was determined by rectal palpation between Day 40 and Day 90 and differences between treatments were analyzed by chi-square. The development of the CVM has enabled the successful cryopreservation of bovine IVP embryos. In the laboratory the typical survival (90% re-expansion) and development (80% hatching) of IVP embryos post-vitrification is high and, as demonstrated here, the pregnancy rates after transfer are approaching commercially acceptable levels. However, further research is required to identify factors that may influence success under full field conditions, for both the IVP and the vitrification technologies. Table 1. Pregnancy rates for fresh IVP, CVM-vitrified IVP, and traditionally flushed embryos cryopreserved in glycerol