Open Pulled Straw Vitrification Research Articles

Effects of different cryopreservation methods on canine isolated preantral follicles

The aim of the present study was to compare the survival and developmental rate of canine isolated preantral follicles (PAFs) after cryopreservation with different methods (closed vs open vitrification). Follicles were isolated from ovaries randomly divided into three groups: fresh control, OPS (open pulled straw) vitrified and cryotube (CT) vitrified. Post-thaw viability of follicles and oocytes was assessed. Fresh and vitrified/thawed PAFs were cultured in 20 µl drops of FSH-supplemented medium for 10 days. Follicular growth, survival rate, estradiol production and ovulation rate were examined. CT method resulted in lower rate of live cells (58.7%) and oocytes (38.8%) than that of fresh ones (83.6% and 64%, respectively) and OPS (80.3% and 79.3%, respectively). Survival rate was similar to fresh follicles in OPS group (98.5% and 95.4%, respectively), while CT decreased the survival to 81.2%. Fresh follicles showed continuous growth, while CT follicles stopped to increase their size after 2 day. In the OPS vitrified follicles, this halting occurred between Day5 and Day10. Fresh follicles showed the highest estradiol production (range: 26.9 – 266.2 pg/ml). Comparing the two vitrified groups, lower estradiol concentration range was measured in the CT group (7.8–48.7 pg/ml vs. 15.4–89.6 pg/ml). Ovulation rate in each group was lowest in the OPS group (1.7% vs 7% and 8.9% in fesh and CT, respectively). Our data show that OPS vitrification provides superior survival rate, in vitro growth and hormonal production to CT. To our knowledge, these are the first results on comparing different cryopreservation protocols on canine isolated preantral follicles.

Ultrastructural changes in immature ovine cumulus-oocyte complexes vitrified in conventional and open pulled straws

This study aimed to evaluate the effect of different cryodevices [conventional straws (CS) and open pulled straws (OPS)] of vitrification on viability, morphology and ultrastructure of ovine immature oocytes. Ovaries of slaughtered ewes were transported from a local abattoir to our laboratory in 0.9 % normal saline at 25–30 °C. The visible follicles (1–6 mm in diameter) were sliced with a microblade and their cumulus-oocyte complexes (COCs) were released in Dulbecco’s phosphate buffer saline. Upon examination under a stereomicroscope, good quality oocytes (n = 266) were selected and randomly assigned into three groups: 1) Fresh oocytes (control, n = 78); 2) Oocytes vitrified using CS (n = 95) and 3) Oocytes vitrified using OPS (n = 93). The gross morphology of COCs was evaluated under a stereomicroscope whilst their viability was evaluated in Trypan blue stained smear. Moreover, glutaraldehyde-fixed COCs (n = 45) were processed for transmission electron microscopy. It was observed that the proportion of recovered normal ovine oocytes after vitrification in OPS (89.24 %) was significantly higher (P < 0.05) than those vitrified in CS (73.68 %). Meanwhile, the proportion of abnormal oocytes was greater (16.84 %) in CS than OPS (8.60 %). The proportion of viable ooplasm and viable cumulus oocytes was significantly (P < 0.05) higher in the fresh group (88.88 %) than OPS (69.11 %) and CS (54.54 %) groups with significant (P < 0.05) difference between the two vitrification groups. Notably, ultrastructural alterations of vesicles and lipid droplets in fresh oocytes were similar with OPS and lower than CS vitrified-warmed oocytes without significant difference between the two vitrified groups. In conclusion, OPS vitrified-warmed oocytes have less cryoinjuries and higher survivability in comparison with CS vitrified-warmed oocytes, although ovine immature oocytes were more vulnerable to cryopreservation by vitrification.

Thin-Film Evaporation Heat Transfer of Liquid Nitrogen and Its Application in Cell Vitrification

Abstract Cell vitrification has been an important method of cell cryopreservation. The faster the cooling rate is, the higher the cell survival rate is. However, in conventional cell vitrification methods, film boiling forms a vapor-blanket on the surface, which hinders further improvement of the cooling rate. To eliminate the problem, this article attempted to replace film boiling with thin-film evaporation (TFE) of liquid nitrogen. The experimental system was developed to investigate the TFE heat transfer characteristics of liquid nitrogen. Then, prostate cancer cells were cryopreserved by TFE vitrification method, open pulled straw vitrification method, and equilibrium freezing method. The results showed that the vitrification method of TFE obtained a higher cooling rate and better cell survival rate than the two other cell cryopreservation methods. Thus, the feasibility of this method was preliminarily proved viable when applied to the cell vitrification process. In addition, both the cooling rate and the cell survival rate are affected by the concentration of the cryoprotectant in the cell suspension. The cooling rate decreases as the concentration of the cryoprotectant increases, but the cell survival rate increases first and decrease afterward with an increase in the cryoprotectant concentration, in which an optimum value exists. This study demonstrates the practicality of the new ultrafast cell vitrification method.

A Rho-Associated Coiled-Coil Containing Kinase Inhibitor, Y-27632, Improves Viability of Dissociated Single Cells, Efficiency of Colony Formation, and Cryopreservation in Porcine Pluripotent Stem Cells.

The establishment of porcine epiblast stem cells (pEpiSCs) and induced pluripotent stem cells (piPSCs) derived from diametrical derivations is of great importance in developing biomedical models. However, pEpiSCs and piPSCs have been technically much harder to culture than mouse embryonic stem cells, showing problematic properties such as spontaneous differentiation and apoptosis after cryopreservation. Therefore, we demonstrated that Y-27632 as a Rho-associated coiled-coil containing kinase inhibitor could prevent dissociated pEpiSCs and piPSCs from undesirable differentiation and apoptosis in cryopreservation protocols. pEpiSC 2, 8 lines, Sendai virus-induced pluripotent stem cells (Sev-iPSCs), and lentivirus-induced pluripotent stem cells were cultured with 10 μM Y-27632 before collecting dissociated cells retrieved from colonies using various enzymes. Dissociated single cells were transferred into freezing mediums (open pulled straw vitrification, STEM-CELLBANKER® (SCB), 10% dimethylsulfoxide in serum) for cryopreservation. The rates of viability and colony formation obtained from dissociated porcine stem cells after freezing/thawing were examined in the presence of Y-27632. The characteristics of pluripotency and in vitro differentiation were also examined in these stem cells treated with Y-27632 after cryopreservation. As a result, the viability and efficiency of colony formation of dissociated pEpiSCs (2, 8 lines) and Sev-iPSCs treated with 10 μM Y-27632 using the SCB cryopreservation protocol were significantly increased when compared with those of nontreated Y-27632 (p < 0.05). Pluripotency genes (OCT-3/4, NANOG, and SOX2) were positively expressed in Y-27632-treated porcine pluripotent stem cells. Also, in vitro differentiation of these stem cells was successfully induced in the presence of 10 μM Y-27632. These results indicated that treatment of Y-27632 for single-cell dissociation and the SCB cryopreservation protocol could facilitate handling porcine pluripotent stem cells and provide the widespread use of these stem cells.

AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring.

BackgroundThe open pulled straw (OPS) vitrification method has been successfully applied in mouse, pig, and goat embryos as well as in buffalo oocytes, but it has not yet been applied to the microinjected embryos. This study examined the effects of OPS vitrification on embryo development and the reproductive capacity of the transgenic offspring in order to establish a method for preservation of microinjected embryos.MethodsOvine pronuclear embryos were microinjected with the exogenous aralkylamine N-acetyltransferase gene (AANAT), frozen by the OPS method, and subsequently thawed for embryo transplantation. Pregnancy rate, lambing rate, survival rate, average birth weight and transgenic positive rate as well as reproduction efficiency and hormone level of the transgenic offspring were investigated to analyze the effect of OPS vitrification on microinjectd pronuclear embryos.ResultsNo significant differences were observed in the birth rate, lamb survival rate and transgenic positive rate between the frozen and non-frozen AANAT-microinjected pronuclear embryos. The average birth weight of the frozen embryos offspring was greater than that of the non-frozen embryos. Importantly, the transgenic offspring that overexpressed the AANAT gene showed improved ovulation efficiency and lambing rate by regulating their hormone levels.ConclusionsThe OPS vitrification approach may be a valuable method in microinjected- embryo transfer technology, which could reserve embryos and result in fewer unnecessary animal sacrifices. In addition, the AANAT+ transgenic offspring exhibited improved reproductive capacity on account of regulation effect of melatonin on reproductive hormone. These data may provide available references for human-assisted reproduction.

High Pregnancy and Calving Rates with a Limited Number of Transferred Handmade Cloned Bovine Embryos.

A major obstacle of widespread commercial application of bovine somatic cell nuclear transfer is the low overall efficiency, that is, healthy calf-late pregnancy per transferred embryo rate. In this study, we report a series of experiments with a limited number of embryos created with handmade cloning (HMC) and transferred without or after open pulled straw vitrification. Embryo reconstruction was performed by using in vitro matured oocytes and adult ear skin fibroblasts. In two experiments, a total of 53 D7 blastocysts were developed from 188 reconstructed embryos. Fresh transfer of seven blastocysts into six recipients has resulted in three early pregnancies, two of them developed over 90 days and eventually resulted in healthy calves (33% offspring/transfer rate). In another two experiments, a total of 11 D7 blastocysts were obtained from 36 reconstructed embryos. Out of these, eight have reexpanded 18 hours after vitrification and warming. Transfer of these blastocysts into eight recipients has resulted in four early pregnancies and two live births; 25% offspring/transfer rate. These results indicate that low overall efficiency may not be an intrinsic feature of cattle cloning, and selection of the right procedures may help to overcome the actual limitations.

Cytoskeleton Genes Expression and Survival Rate Comparison Between Immature and Mature Yak Oocyte After OPS Vitrification

ABSTRACTThis study was conducted to investigate the effect of vitrification on survival rate and cytoskeleton gene expression during yak oocyte maturation. The yak oocytes were incubated for 0 h [germinal vesicle (GV) stage] and in vitro matured for 24 h [metaphase II (MII) stage] to obtain immature and mature oocytes. Survival rate after vitrification were compared between immature and mature yak oocytes and cytoskeleton-related genes [cytokeratin 8 (CK8), β-actin (ACTB), and gap junction protein, alpha 1 (GJA1)] were tested by real-time PCR. Our results showed that MII stage survival rate after open pulled straw vitrification (35.60%) is significantly higher than GV stage (25.90%) oocytes. Furthermore, expression of CK8, ACTB, and GJA1 in MII stage oocytes are also significantly higher than GV stage oocytes. In conclusion, our study demonstrated that higher expression of GJA1, CK8, and ACTB in vitrify-warmed MII stage oocytes when compared with GV stage oocytes and such discrepancy might result in higher survival rate in vitrify-warmed MII stage oocytes.

Changes in mitochondrial function in porcine vitrified MII-stage oocytes and their impacts on apoptosis and developmental ability

The purpose of this study was to investigate the changes in mitochondria in porcine MII-stage oocytes after open pulled straw (OPS) vitrification and to determine their roles in apoptosis and in vitro developmental ability. The mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) level, adenosine-5′-triphosphate (ATP) concentration, mitochondrial distribution, mitochondrial ultrastructure, early-stage apoptosis with Annexin V-FITC staining, survival rate, parthenogenetic developmental ability and related gene expression were measured in the present experiments. The results showed that: (1) the mitochondrial ΔΨm of vitrified-thawed oocytes (1.05) was lower than that of fresh oocytes 1.24 (P<0.05). (2) ROS level in the OPS vitrification group was much higher than that of the fresh group, while the ATP concentration was much lower than that of fresh group (P<0.05). (3) Early-stage apoptosis rate from the OPS vitrification group (57.6%) was much higher than that of fresh group (8.53%) (P<0.05), and the survival rate and parthenogenetic cleavage rate of OPS vitrified oocytes were much lower than those from fresh ones (P<0.05). (4) Vitrification not only disrupted the mitochondrial distribution of porcine MII-stage oocytes, but also damaged the mitochondrial ultrastructure. (5) After vitrification, the gene expression level of Dnm1 was up-regulated, and other four genes (SOD1, Mfn2, BAX and Bcl2) were down-regulated. The present study suggested that not only the morphology and function of mitochondria were damaged greatly during the vitrification process, but also early-stage apoptosis was observed after vitrification. Intrinsic mitochondrial pathway could be in involved in the occurrence of apoptosis in vitrified-thawed porcine oocytes.

Effective cryopreservation of golden Syrian hamster embryos by open pulled straw vitrification.

Golden Syrian hamster embryos are difficult to cryopreserve due to their high sensitivity to cryoprotectants and in vitro handling. The objective of this study is to develop a robust open pulled straw (OPS) vitrification technique for cryopreserving hamster embryos at various developmental stages. We first systematically tested the concentrations of cryoprotectants and the exposure times of two-cell embryos to various vitrification solutions. We identified pretreatment of two-cell embryos with 10% (v/v) ethylene glycol (EG) + 10% (v/v) dimethylsulfoxide (DMSO) for 30 s followed by exposure in the vitrification solution, EDFS30 (containing 15% EG + 15% DMSO), for 30 s before plunging into liquid nitrogen (two-step exposure method) as the optimal OPS vitrification protocol. We then investigated the resourcefulness of this protocol for vitrifying hamster embryos at different developmental stages. The results showed that high blastocyst rates from embryos vitrified at two-cell, four-cell, eight-cell, or morula stage (62%, 78%, 80%, or 72%, respectively), but not those verified at pronuclear (0%) or blastocyst stage (24%; P < 0.05), were achieved by this protocol. When embryos vitrified at the two-cell stage were recovered and then directly transferred to recipient females, 29% of them developed to term, a development rate not significantly different (P > 0.05) from the 40% birth rate of the unvitrified controls. In conclusion, we have developed an effective two-step OPS vitrification protocol for hamster embryos.

Open pulled straw vitrification and slow freezing of sheep IVF embryos using different cryoprotectants.

The aim of the present study was to evaluate the post-thaw survival and hatching rates of sheep blastocysts using different cryoprotectants. In Experiment 1, Day 6 sheep embryos were cryopreserved by a slow freezing protocol using 10% ethylene glycol (EG), 10% dimethyl sulfoxide (DMSO) or a mixture of 5% EG and 5% DMSO. Hatching rates were higher in the 10% EG group than in the 10% DMSO or EG + DMSO groups (30% vs 18% and 20%, respectively). In Experiment 2, embryos were cryopreserved by open pulled straw (OPS) vitrification using either 33% EG, 33% DMSO or a mixture of 16.5% EG + 16.5% DMSO. Re-expansion and hatching rates in the EG + DMSO group (79.16% and 52.74%, respectively) were higher than those in the EG group (64.28% and 30.02%, respectively), whereas the outcomes for the DMSO group were the lowest (45.18% and 8.6%, respectively). In Experiment 3, embryos were cryopreserved by OPS vitrification using either 40% EG, 40% DMSO or a mixture of 20% EG + 20% DMSO. Re-expansion and hatching rates were highest in the EG group than in the EG + DMSO and DMSO groups (92.16% vs 76.30% and 55.84% re-expansion, respectively; and 65.78% vs 45.55% and 14.46% hatching, respectively). In conclusion, OPS vitrification was found to be more efficient for cryopreservation of in vitro-developed sheep embryos than traditional freezing.

Production of Wild Buffalo (Bubalus arnee) Embryos by Interspecies Somatic Cell Nuclear Transfer Using Domestic Buffalo (Bubalus bubalis) Oocytes

The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin-18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open-pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re-expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified-warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.

Survivability of caprine oocytes vitrified in conventional and open pulled straws

The aim of this study was to compare the efficacy of conventional straw vitrification with open pulled straw vitrification in terms of cryosurvivability and damages caused to the oocytes. Two hundred immature goat cumulus oocyte complexes (COCs) were vitrified in a solution of ethylene glycol, dimethyl sulfoxide, and sucrose using either conventional straws (100 COCs) or open pulled straws (100 COCs). The COCs were warmed and observed for morphological damages and viability after 7 days of preservation in liquid nitrogen. Among the 100 COCs cryopreserved in each case, only 83 COCs were recovered after warming in conventional straw vitrification as compared to 94 COCs in open pulled straw vitrification. In terms of morphological survivability, the percentage of morphologically normal oocytes was greater (P < 0.01) in the case of open pulled straw vitrification (86.2%) as compared to conventional straw vitrification (59.0%). Viability percentage of live oocytes was greater (P < 0.01) in the case of open pulled straw vitrification (90.4%) as compared to conventional straw vitrification (66.3%). The results indicate that open pulled straw vitrification is better than conventional straw vitrification for rapid freezing of immature goat COCs in terms of both morphological survival and viability.

47 COMPARISON BETWEEN CRYOLOOP AND OPEN PULLED STRAW VITRIFICATION METHODS FOR BOS INDICUS BLASTOCYSTS

A major obstacle of large-scale commercial application of bovine in vitro fertilization is the lack of a suitable cryopreservation method for supernumerary embryos produced. The traditional slow-freezing method has proven to be effective for embryos of a wide range of mammalian species; however, the formation of intracellular ice is still a challenge and the efficiency needs to be improved. Over the past decade, several advances have taken place in vitrification technologies, such that it can provide high efficiency with better pregnancy outcome due to its high cooling rates and the lack of crystals formed inside the cells. Most vitrification methods have been evaluated in Bos taurus cattle but more still remains to be investigated in Bos indicus races predominant in the tropics. There are several vitrification protocols and holders, including CryoLoop, open pulled straw (OPS), MS Grids, and Cryotop, among others. The CryoLoop method uses a nylon loop attached to a metal Cryovial lid were blastocysts are placed on an equilibration solution film. CryoLoop cooling rates are approximately 20.000°C min–1 and have shown very good results in humans. The OPS is a well-known support for bovine blastocysts; the embryos are taken by capillarity into the OPS and use a 1- to 2-μL drop of final equilibration solution. Cooling rates using this method are approximately 2.000°C min–1. The aim of this work was to prove CryoLoop and OPS vitrification methods in Bos indicus blastocyst and compare re-expansion and hatching rates 24 h after warming. Ovaries were collected from a local slaughterhouse and cumulus-oocyte complexes (COC) were treated for the standard IVF method. A total of 60 blastocysts were vitrified in CryoLoops and 68 blastocysts in OPS (within 4 repeats). For CryoLoops, groups of 2 blastocysts were placed in a solution of 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 3 min, and then were placed in a solution of 15% EG, 15% DMSO, 10 mg mL–1 of Ficoll 70, and 0.65 M sucrose for 20 s, and rapidly were put into the nylon loop and taken to the LN. For OPS, groups of 2 to 3 blastocysts were placed in a solution of 10% EG and 10% DMSO for 1 min, and then were placed in a solution of 20% EG and 20% DMSO for 20 s, and rapidly were taken by capillarity into the OPS and taken to the LN. Thawing was the same for both treatments; vitrified blastocysts were taken out from the LN and rapidly put into a solution of 0.3 M sucrose for 2 min and then put into a solution of 0.2 M sucrose for 3 min, were washed twice in TCM199 supplemented with 10% FCS, and cultured for 24 h in CR1aa media. Data were analysed using the R language. Media comparison for proportions was done using a chi-squared test. No significant difference was observed in re-expansion or hatching rates between CryoLoop and OPS supports (P = 0.01 for both); however, the CryoLoop method showed more efficiency than OPS in re-expansion rate (65 v. 44.4%, respectively) and hatching rate (30.8 v. 20%, respectively). In all cases, the CryoLoop method showed much better outcomes. The results indicate that vitrification in CryoLoops is a suitable method for cryopreservation of Bos indicus blastocysts.

Cytokeratin distribution and expression during the maturation of mouse germinal vesicle oocytes after vitrification

Cytokeratin (CK) is a type of the cytoskeleton that increases cell stabilization during oocyte maturation. This study was conducted to investigate the effect of vitrification on the distribution and expression of CK during mouse oocyte maturation. Germinal vesicle (GV) oocytes were randomly allocated into three groups: (1) untreated (fresh), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (CPA exposure), or (3) vitrified using the open-pulled straw (OPS) method (vitrification). The oocytes were then incubated for 0h, 6h (metaphase I (MI) stage), and 10h (metaphase II (MII) stage). The CK distribution in the oocytes at the GV, MI, and MII stages was observed by immunofluorescence, and the expression at the MII stage oocytes derived from vitrified GV oocytes was detected by Western blotting. The CK distribution in the GV oocytes (88.5%) displayed a cortical pattern in the fresh group, whereas a granular pattern was mainly found at the MI (86.7%) and MII (93.5%) stages. In the CPA exposure group, 90.3% of the GV oocytes were observed to display the cortical pattern, and 69.2% of the MI oocytes and 92.9% of the MII oocytes showed the granular pattern. In the vitrification group, most oocytes (GV, 88.9%; MI, 100%; MII, 93.3%) exhibited the cortical pattern. The CK fluorescence intensities of the MII stage oocytes in both the fresh (59.27) and CPA exposure (60.05) group were significantly higher than that of the vitrification (26.53) group (p<0.05). Western blotting showed that the CK expression in the MII oocytes derived from vitrified GV oocytes was significantly lower (p<0.05) than in the control. In conclusion, OPS vitrification affects the normal CK distribution pattern during oocyte maturation and results in decreased CK expression in MII oocytes derived from vitrified GV oocytes.

Open pulled straw vitrification of in vitro matured sheep oocytes using different cryoprotectants

The aim of this study was to compare the survival and in vitro development of sheep oocytes after open pulled straw vitrification and different final concentrations of permeable cryoprotectants. In 5 identical replicates of two experiments, in vitro matured (IVM) oocytes were vitrified by the Open Pulled Straw (OPS) method, then warmed, and the surviving ones were subjected to parthenogenetic activation. In Experiment 1, survival rate of oocytes after vitrification in 33% ethylene glycol was higher than in 33% DMSO or a mixture of 17.5% ethylene glycol and 17.5% DMSO (87.64 vs. 77.43 vs. 69.39%, respectively). The cleavage and blastocyst rates were higher after vitrification in mixture group than in ethylene glycol and DMSO (46.81 and 15.5 vs. 37.55 and 9.12 vs. 29.51 and 6.40%, for cleavage and blastocyst rates in different groups, respectively). In Experiment 2, elevated concentrations of vitrification solutions were used. The survival rate was higher after vitrification in 40% ethylene glycol and in the mixture of 20% ethylene glycol and 20% DMSO than in 40% DMSO (90.22 vs. 87.56 vs. 75.34%, respectively). Cleavage and blastocyst rates were also higher in the ethylene glycol and ethylene glycol – DMSO mixture group than in DMSO alone group (50.67 and17.60 vs. 49.13 and 14.45 vs. 33.86 and 9.81% for cleavage and blastocyst rates in different groups, respectively). The survival rates between the two experimental groups was higher in 40% ethylene glycol group, 40% mixture group and 33% ethylene glycol group than in 40% DMSO group, 33% mixture group and 33% DMSO group. Cleavage and blastocyst rates were higher in 40% ethylene glycol group, 40% mixture group and 33% mixture group than in 40% DMSO group, 33% ethylene glycol group and 33% DMSO group. All cleavage and blastocyst rates in both the experiments were lower than those of the non-vitrified control group (87.00 and 45.00, respectively). In conclusion, although ethylene glycol group and ethylene glycol – DMSO mixture group gave better survival and cleavage – blastocyst rates than DMSO group, the survival rates were lower than the control group and hence the technique could be further improved to get better results after OPS vitrification of IVM sheep oocytes.

Cryopreservation of Zona‐Free Cloned Buffalo (Bubalus Bubalis) Embryos: Slow Freezing vs Open‐Pulled Straw Vitrification

This study was carried out to compare the post-thaw cryosurvival rate and the level of apoptosis in vitro produced zona-free cloned buffalo blastocysts subjected to slow freezing or vitrification in open-pulled straws (OPS). Zona-free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re-expansion rate following post-thaw culture for 22-24h. The post-thaw re-expansion rate was significantly (p<0.05) higher following vitrification in OPS (71.2±2.3%) compared with that after slow freezing (41.6±4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen-thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non-cryopreserved cloned embryos (422.6±67.8) and those cryopreserved by slow freezing (376.4±29.3) or vitrification in OPS (422.8±36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8±2.0) or OPS vitrification (13.3±1.8), was significantly (p<0.05) higher than that for the control non-cryopreserved cloned embryos (3.4±0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona-free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.

Cryopreservation of human testicular diploid germ cell suspensions

For patients with threatened fertility, preservation of it is a major concern. Although promising results have been obtained in animal models using testicular germ cell suspensions, in humans, it is crucial to first develop an efficient method of cryopreservation to be able to apply to transplantation. Thus, four reliable and available cryopreservation techniques in any fertility centre were tested to cryopreserve an enriched fraction of diploid germ cells isolated from human testicular biopsies. The protocols were evaluated based on cell viability, and the results showed significant differences between the four methods. The semen and tissue cryopreservation methods appeared to be inadequate for diploid germ cell suspensions, and programmed slow freezing gave significantly lower results than open pulled straw vitrification; the latter was found to be the protocol that best preserved cell viability. The vitrification of isolated human diploid germ cells is innovative and constitutes valuable information for cryopreservation in cases of transplants or in vitro maturation.

Controlled Freezing and Open-Pulled Straw (OPS) Vitrification of In vitro Produced Bovine Blastocysts Following Analysis of ATP Content and Reactive Oxygen Species (ROS) Level

To our knowledge, no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT) by controlled freezing and vitrification. This experiment, therefore, was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification. Adenosine-5′-triphosphate (ATP) content and reactive oxygen species (ROS) level in blastocysts were also analyzed. Firstly, for each type of blastocyst (IVF, ICSI or SCNT), significant differences were observed between the survival rates of the controlled freezing ((81.56±2.33), (68.18±4.72) or (47.89±5.83)%) and OPS vitrification groups ((92.24±4.54), (82.40±3.76) or (78.71±5.91)%; P<0.05). Secondly, for each type of blastocyst (IVF, ICSI or SCNT), ATP content was significantly decreased after controlled freezing or vitrification, and the ATP content in the controlled freezing group (0.43±0.06), (0.35±0.05) or (0.21±0.02) pmol) was significantly lower than that found in the OPS vitrification group (0.62±0.04), (0.46±0.03) or (0.30±0.01) pmol; P<0.05). Thirdly, ROS level in fresh IVF ((47.33±3.56) c.p.s (counted photons per second), ICSI ((36.51±2.58) c.p.s) or SCNT blastocysts ((26.44±1.49) c.p.s) was significantly lower than that found in the OPS vitrification group ((72.14±4.31), (58.89±3.89) or (40.11±5.73) c.p.s; P<0.05), but higher than that of the controlled freezing group (34.41±3.32), (23.13±1.26) or (15.46±2.45) c.p.s; P<0.05). The present study indicated that vitrification is more efficient in the cryopreservation of bovine blastocysts derived from IVF, ICSI or SCNT than controlled freezing. Furthermore, both vitrification and controlled freezing significantly altered the ATP content and ROS level in those blastocysts.

Cryopreservation of immature equine oocytes, comparing a solid surface vitrification process with open pulled straws and the use of a synthetic ice blocker

The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N = 269), the OPS (N = 159) and the SSV (N = 202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P < 0.001) and MIn (76.6 vs. 31.1 and 33.7%; P < 0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in the OPS vitrification process increased the rates of both IVM (30.5%; P < 0.01) and MIn (45.8%; P < 0.05) of the oocytes (N = 59). Including 0.1% ice blocker in the SSV process improved the IVM rate (20.9%; P < 0.05), whereas MIn remained compromised in this group (N = 67). However, increasing the concentration of the ice blocker (to 1.0%) in the cryopreservation methods did not significantly improve rates of IVM. In conclusion, the addition of a synthetic ice blocker (0.1%) to both cryopreservation processes significantly increased rates of both IVM and MIn of immature equine oocytes cryopreserved by OPS.

72 SUPERIOR PREGNANCY RATES WITH THE TRANSFER OF OPEN PULLED STRAW-VITRIFIED V. CONVENTIONALLY CRYOPRESERVED EMBRYOS IN GOATS

Two separate studies were conducted in the course of which goat embryos were cryopreserved either by the open pulled straw (OPS) procedure of (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58) or by conventional slow freezing. Embryos of each doe were assigned to both methods. A total of 94 morphologically intact blastocysts, collected by transcervical flushing from superovulated pluriparous Boer goat does, were involved. For OPS-vitrification the method of Vajta et al. (1998) was slightly modified (El-Gayar et al. 2008 Cryobiology 57, 191–194). It involves exposure of the embryos to a medium containing 10% dimethyl-sulfoxide (Me2SO) + 10% ethylene glycol (EG) for 1 min, followed by 20 s in medium containing 20% Me2SO + 20% EG. After aspiration into thinly drawn-out straws, the straws were plunged directly into liquid nitrogen. Conventional freezing comprises equilibration in M2 medium containing 1.4 M glycerol and cooling in 0.25-mL straws at a rate of 0.3°C min–1 as described in (Nowshari and Holtz 1993 J. Anim. Sci. 71, 3403–3408). Embryos of both treatment groups were transferred to synchronized recipients (2/recipient) within 2 h after thawing. The surrogate mothers were diagnosed for pregnancy 5 weeks later and, if gravid, permitted to carry to term. Differences between means were tested for significance by the chi-square test. All embryos were recovered after thawing. Within the 2-h period from thawing to transfer, OPS-vitrified embryos had the appearance of flawless, expanded blastocysts, whereas conventionally frozen–thawed embryos had not quite reached full re-expansion. Of 25 recipients receiving OPS-vitrified blastocysts, 22 (88%) kidded with average litter size of 1.5. Of 22 recipients receiving conventionally frozen–thawed blastocysts, 10 (45%) kidded with average litter size of 1.8. Embryo survival in vitrified blastocysts amounted to 67%, and in conventionally frozen–thawed blastocysts 42%. The difference in kidding rate between the two methods was statistically significant (P &lt; 0.01). Regardless of the cryopreservation method all kids born were viable, their birth weight being within the range typical for the population. The results indicate that, for the cryopreservation of caprine blastocysts, OPS vitrification is a more suitable technique than conventional freezing and should be routinely employed. The senior author owes thanks to the Arab Fund for Economic and Social Development for the financial support.