Open Pulled Straw Research Articles

Ultrastructure of bovine oocytes exposed to Taxol prior to OPS vitrification

Our objective was to document potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the open pulled straw (OPS) method. Oocytes were divided into four experimental groups for cows and four groups for calves: (1) a control group fixed immediately after maturation; (2) an OPS group cryopreserved by conventional OPS; (3) a Taxol/CPA group exposed to 1 microM Taxol and cryoprotective agents (CPAs); and (4) a Taxol/OPS group vitrified by OPS including 1 microM Taxol to the vitrification solution. All oocytes were processed for light and transmission electron microscopy. The main injuries were observed on the metaphase plate and the spindle. In control oocytes, the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules in both cow and calf oocytes. However, in cow OPS oocytes, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized as typical spindles. In contrast, cow Taxol/OPS oocytes as well as both cow and calf Taxol/CPAs oocytes showed well-organized metaphase plates and normal spindle morphology. All calf OPS and calf Taxol/OPS oocytes displayed a single block of chromatin and no microtubules could be observed around the chromosomes. In conclusion, treatment with 1 microM Taxol before and during vitrification did not induce adverse changes in the oocyte cytoplasm or metaphase spindles in adult bovine oocytes, but stabilized the metaphase and spindle morphology.

Bulk vitrification of human embryonic stem cells

The traditional vitrification method cannot keep up with the increased culture and propagation efficiency required to cryopreserve large quantities of vigorously proliferating human embryonic stem (HES) cells. In this study, we describe a newly invented vitrification carrier for cryopreserving large amount of HES cells and evaluate whether this bulk vitrification (BV) method is as effective as the popular open-pulled straw (OPS) vitrification method. HES cell clumps were harvested after passage and transferred to a cell strainer; only those clumps with a diameter more than 70 microm were included in the study and randomly selected to be cryopreserved by the BV method, OPS vitrification or slow freezing method. HES cell survival, growth and pluripotency were analyzed after thawing. Bulk vitrification method with cell strainer could cryopreserve 136 +/- 23.4 cell clumps at one time (round), which was 30 times as high as those for OPS method (4 +/- 1.5). After thawing, bulk-vitrified HES cells exhibited high survival rate up to 94.3%, comparable with the OPS method. All surviving cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of post-thawed, bulk-vitrified HES cell clumps, confirming pluripotency. This new BV method could cryopreserve a large quantity of HES cell clumps at one time, which not only would satisfy routine cryopreservation of HES cell during daily culture process but also guarantee researchers have large quantity of efficiently cryopreserved HES cells ready for a scheduled study at any time.

Factors affecting the success rate of porcine embryo vitrification by the Open Pulled Straw method

The objectives of this study were: (1) to evaluate the influence of porcine embryo developmental stage on in vitro embryo development after vitrification, (2) to study the efficiency of the one-step dilution procedure, compared with conventional warming, for vitrified embryos at different stages of development, and (3) to determine the influence of the embryo donor on the in vitro survival of vitrified embryos at morulae and blastocyst stages. Two to four cell embryos, morulae and blastocysts were collected by laparotomy from weaned crossbred sows ( n = 55). Vitrification and conventional warming were performed using the OPS procedure with Superfine Open Pulled Straws (SOPS). For one-step dilution, embryos were placed in 800 μl TCM199-HEPES containing 20% of new born calf serum and 0.13 M sucrose for 5 min. To evaluate development, two to four cell embryos, morulae and blastocysts were cultured in vitro for 120, 48 and 24 h, respectively. Some fresh embryos from each developmental stage were not vitrified and cultured as controls. Embryos were morphologically evaluated for their developmental capacity during the in vitro culture by stereomicroscopy. The total cell number of embryos was assessed by Hoechst-33342 staining and fluorescence microscope observation. There was a significant effect of the stage of development on the in vitro survival, perihatching rate and the number of cells of embryos after vitrification and warming (Experiment 1; p < 0.001). The survival and perihatching rates of two to four cell embryos were lower than those obtained for morulae and blastocysts ( p < 0.001). No differences ( p > 0.05) in survival rates were found between vitrified and fresh blastocysts. The warming procedure did not affect the development and total cell number of vitrified two to four cell embryos, morulae or blastocysts (Experiment 2). However, donor had a significant effect ( p < 0.001) on the in vitro development and the number of cells of morulae and blastocysts after vitrification and warming (Experiment 3). In conclusion, the embryo developmental stage and the embryo donor were important factors that affected the development of porcine embryos after OPS-vitrification and warming. OPS-vitrification and the one-step dilution are efficient procedures to be used with intact porcine morulae and blastocysts.

Piezo-actuated zona-drilling improves the fertilisation of ops vitrified mouse oocytes

The present study was designed to investigate fertilisation of open pulled straw (OPS) vitrified mouse oocytes drilled with piezo-micromanipulation method and their subsequent in vitro and in vivo developmental capacity. Ovulated mouse oocytes were vitrified using the OPS method. After warming, the zona pellucida of a group of vitrified-warmed oocytes was drilled by piezo-micromanipulation. Groups of (a) vitrified, (b) vitrified/drilled and (c) fresh control oocytes were fertilised in vitro. The fertilisation rate of vitrified-warmed oocytes was significantly lower than that of fresh oocytes (45.0 +/- 12.6% vs. 85.2 +/- 6.8%, P < 0.05), and was significantly improved by zona-drilling (85.4 +/- 7.3%). However, blastocyst formation rates of the vitrified and vitrified/drilled groups were significantly lower than those of the fresh controls (65.7 +/- 7.0% and 66.4 +/- 2.5% vs. 86.6 +/- 4.3%, respectively, P < 0.05). The cell number of blastocysts from the vitrified/drilled or the vitrified group was not different from that of the controls. Embryo transfer resulted in pregnancy in all three groups, but the rate of development to term was lower in the vitrified/drilled or vitrified groups than in the controls (16.6 +/- 0.7% or 36.0 +/- 2.4% vs. 51.3 +/- 2.9%, respectively). Our results demonstrated that zona-drilling with piezo-micromanipulation could improve fertilisation in OPS vitrified mouse oocytes but did not increase the overall number of vitrified oocytes developing to term.

Conventional Freezing, Straw, and Open-Pulled Straw Vitrification of Mouse Two Pronuclear (2-PN) Stage Embryos

Little is known on the cryopreservation of mouse pronuclear (PN) stage embryos. In the present experiment the mouse 2-PN stage embryos were cryopreserved by conventional freezing, straw, or open-pulled straw (OPS) vitrificaiton methods. The conventional freezing solution was 1.5 mol/L ethylene glycol (EG), and vitrification solutions were EFS30 (30% EG, Ficoll, and sucrose), EFS40 (40% EG, Ficoll, and sucrose), EDFS30 (15% EG, 15%dimethyl sulfoxide [DMSO], Ficoll, and sucrose), or EDFS40 (20% EG, 20%DMSO, Ficoll, and sucrose). The blastocyst rate of 2-PN stage embryos cryopreserved by conventional method (30.4%) was lower than those vitrified by straw method with EDFS (56.9% to 69.1%), by OPS method (66.0% to 85.7%), and that of control (80.8%) (P < 0.05). With a given vitrificaiton solution EFS30, EFS40, EDFS30, or EDFS40, the blastocyst rate of embryos vitrified by the OPS method (66.7%, 66.0%, 85.7%, or 76.9%) was higher than that of those vitrified by the straw method (46.8%, 43.8%, 69.1%, or 56.9%) (P < 0.05). When mouse 2-PN-stage embryos were vitrified with EDFS30 by straw or OPS method, the highest blastocyst rate was achieved (69.1% or 85.7%) and was similar to that of the control, respectively. The embryos transfer results revealed that the full-term development of blastocysts derived from 2-PN stage embryos vitrified by OPS method with EDFS30 (19.9%) was similar to that of the control (23.5%), and higher than that of those cryopreserved by conventional freezing (9.3%) (P < 0.05). The present research demonstrates that the OPS method, especially with EDFS30, is more effective in cryopreserving mouse 2-PN embryos.

Effect of using slush nitrogen (SN2) on development of microsurgically manipulated vitrified/warmed mouse embryos

This study evaluated the effect of vitrification using slush nitrogen (SN(2)) on cryopreservation of micromanipulated mouse embryos. The zona pellucida of 4-cell embryos was either left intact or dissected or dissected with biopsy of an intact blastomere. In a second study, a blastomere was destroyed and either removed (removed group) or not removed (remained group) prior to vitrification/freezing. The micromanipulated embryos were equilibrated and loaded into an open pulled straw (OPS), and plunged into liquid nitrogen (LN(2)) or SN(2). When using LN(2) vitrification, recovery and blastocyst formation rates of embryos were lower for zona pellucida-opened and/or blastomere-biopsied embryos compared with zona pellucida-intact embryos. Using SN(2) for vitrification resulted in increased survival and development of vitrified/warmed embryos in both the zona pellucida-opened and blastomere-biopsy groups. Similar results were observed when using embryos with a destroyed blastomere either removed or left remaining before vitrification. However, the number of total and apoptotic cells were similar for both LN(2) and SN(2). In addition, using SN(2) increased the rate of intact recovery and blastocyst formation in warmed hemi-8-cell embryos derived from the same embryo. These results suggest that vitrification using SN(2) is useful in cryopreservation of micromanipulated embryos obtained from a variety of programs, including assisted hatching, preimplantation genetic diagnosis and nuclear transfer.

Development of OPS vitrified pig blastocysts: Effects of size of the collected blastocysts, cryoprotectant concentration used for vitrification and number of blastocysts transferred

Unhatched blastocysts from Large White hyperprolific gilts ( n = 103) were identified, measured and vitrified using the Open Pulled Straw (OPS) technique to evaluate the effects of the collected blastocyst size and cryoprotectant concentrations used for vitrification, and the number of embryos transferred per recipient. Vitrified/warmed blastocyst viability was estimated in vitro, as the percentage of embryos developing after 72 h, and in vivo, on pregnancy Day 30. In the in vitro study, we compared the use of three cryoprotectant concentrations (16.5, 18, or 20% DMSO + 16.5, 18, or 20% EG + 0.4 M sucrose). Survival rates differed significantly between the control (98.3%) and the three cryoprotectant concentrations (67, 62.3, and 57%, respectively). Blastocyst size at vitrification determined the further in vitro development of embryos (26% survival for blastocysts 126–144 μm versus 100% for blastocysts >199 μm). For the in vivo study, blastocysts were vitrified using cryoprotectant concentrations of 16.5 or 18% DMSO + EG and transferred surgically in groups of 20 or 30 per recipient ( n = 40). Recipients were slaughtered on pregnancy D30. No significant differences were detected in gestation rates (50–70%) and embryo survival rates (14.7–25%), although survival was higher ( P = 0.0003) when 20 blastocysts were transferred compared to 30 (24.7% versus 15.5%). Our findings indicate that best results, in terms of subsequent in vivo embryo survival, were achieved after transferring 20 embryos at the blastocyst or expanded blastocyst stage, previously vitrified using cryoprotectant concentrations of 16.5 or 18%.

Vitrification devices affect structural and molecular status of in vitro matured ovine oocytes

We evaluated the effect of three different cryodevices on membrane integrity, tubulin polymerization, maturation promoting factor (MPF) activity and developmental competence of in vitro matured (IVM) ovine oocytes. IVM oocytes were exposed during 3 min to 7.5% DMSO and 7.5% ethylene glycol (EG) in TCM199 and 25 sec to 0.5 M sucrose, 16.5% DMSO and 16.5% EG, loaded in open pulled straws (OPS), cryoloops (CL) or cryotops (CT) and immersed into liquid nitrogen. Untreated (CTR) or exposed to vitrification solutions but not cryopreserved (EXP) oocytes were used as controls. After warming, double fluorescent staining evidenced a lower membrane integrity in vitrified groups compared to the controls (P < 0.01). After in vitro fertilization and culture OPS and CL groups evidenced a lower cleavage rate than CT and controls (P < 0.01) while blastocysts were obtained only in CL and EXP, at a lower rate than CTR (P < 0.01). All vitrified groups showed alterations in spindle conformation, which were partially recovered in OPS and CT groups. MPF activity was lower in treated compared to CTR and CT showed the lowest value (P < 0.01). After 2 hr culture MPF activity was restored in all groups except CT. Parthenogenetic activation was higher in treated compared to CTR and CT evidenced the highest value. Our results indicate that cryodevice influences not only the ability to survive cryopreservation but is also associated with molecular alterations which affect developmental competence.

Open-pulled Straw (OPS) Vitrification of Mouse Hatched Blastocysts

This study was first employed to investigate the developmental potential of mouse hatched blastocyts (HBs) vitrified by a two-step open-pulled straw (OPS) method. HBs were obtained by culture of morulae in vitro. First, the embryos were placed in four cryprotectant solutions—that is, 10% ethylene glycol (EG), 10%E + 10%D (10% EG and 10% dimethyl sulphoxide (DMSO) in mPBS), EFS30 (30% EG, Ficoll, and sucrose) and EDFS30 (15% EG, 15% DMSO, Ficoll, and sucrose)—at 25°C for 0.5 to 10 min, respectively, to determine their optimal survival after rapid dilution in 0.5 M sucrose. Secondly, based on the above best survival, the embryos were plunged into liquid nitrogen after first pretreatment in 10%E for 0.5 min and then 0.5 min equilibration in EFS30 (Group 1), or 10%E + 10%D and EDFS30 for 0.5 min, respectively (Group 2). When warming, three methods were used to dilute the cryoprotectants from the vitrified embryos. The embryos were assessed by the re-expansion of the blastocoel or development to term. The result showed that all the vitrified-warmed HBs got high in vitro survival rates (83.7% to 98.9%). The highest in vitro survival rates (87.8% in Group 1, 98.9% in Group 2) were obtained when the vitrified embryos were diluted first in 0.3 M sucrose for 5 min, then in 0.15 M sucrose for 2 min (method C). When the vitrified embryos diluted with method C were transferred, their survival rate in vivo (35.5% to 42.2% of the total) were similar to (P > 0.05) that of control (45.7%). These results demonstrate OPS method was highly efficient for the cryopreservation of mouse HBs.

Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration

This study was designed to examine the reduced incidence of normal fertilization in vitrified ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In experiment 1, the treated and control oocytes were matured for another 2 h, and the oocytes were then in vitro fertilized for 12 h to examine sperm penetration. The percentage of monospermy in toxicity group (29.3%) and vitrification group (28.2%) dramatically decreased compared to the control group (45.0%) ( P < 0.05). To find the mechanism that the VS decreased the monospermy, some treated and control oocytes were used to test the distribution of CG and the resistance of zona pellucida (ZP) to 0.1% pronase E immediately (IVM 24 h), after another 2 h of maturation (IVM 26 h), and after 12 h of in vitro fertilization (IVF 12 h) respectively. Others were used to examine female pronucleus formation after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the percentage of CG completely release in the oocytes (IVM 24 and 26 h) of toxicity group (41.2% and 39.9%) and vitrification group (41.7% and 51.7%) was significantly higher than that of control group (7.1% and 18.4%) ( P < 0.05). The ZP digestion duration in the oocytes (IVM 26 h) of the toxicity group (435.6 s) and vitrification group (422.3 s) was longer than that of control group (381.6 s) ( P < 0.05). The percentage of female pronucleus formation in toxicity group (58.7%) and vitrification group (63.9%) was higher than that (8.2%) of control group ( P < 0.05). The data above demonstrated that the VS containing DMSO and EG could parthenogenetically activate in vitro matured ovine oocytes, resulting in ZP hardening and decreased sperm penetration.

Vitrification of Boer Goat Morulae and Early Blastocysts by Straw and Open‐Pulled Straw Method

The aim of this study was to investigate the effects of different vitrification solutions [EFS30 or EFS40 contains 30% (v/v) ethylene glycol (EG), 40% (v/v) EG; EDFS30 or EDFS40 contains 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) EG and 20% (v/v) DMSO], equilibrium time during vitrification (0.5-2.5 min) and vitrification protocols [one-step straw, two-step straw and open-pulled straw (OPS)] on in vivo development of vitrified Boer goat morulae and blastocysts after embryo transfer. In the one-step straw method, the lambing rates of vitrified embryos in EFS30 (37.5%), EFS40 (40.5%) or EDFS30 (38.2%) group were similar to that of fresh embryos (57.5%) and conventional freezing method (46.7%) when the equilibrium time was 2 min. In the two-step straw method, the highest lambing rate was obtained when embryos were pretreated with 10% EG for 5 min and then exposed to EFS40 for 2 min (51.4%), showing similar lambing rates compared with fresh embryos (56.1%) or the embryos cryopreserved by conventional freezing method (45.2%). In the OPS method, the lambing rate in EFS40, EDFS30 or EDFS40 groups were similar to that (57.1%) of fresh embryos, or to that (46.0%) of embryos cryopreserved by conventional freezing method. The highest lambing rate (51.4%) of the group of OPS was obtained when the embryos were vitrified with EDFS30. In conclusion, either the two-step straw method in which embryos were pretreated in 10% EG for 5 min and then exposed to EFS40 for 2 min, or the OPS method in which embryos were pretreated in 10% EG + 10% DMSO for 30 s and then exposed to EDFS30 for 25 s was a simple and efficient method for the vitrification of Boer goat morulae and blastocysts.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P &lt; 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P &lt; 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

89 IMPROVED POST-WARMING DEVELOPMENTAL COMPETENCE OF OPEN PULLED STRAW-VITRIFIED IN VITRO-PRODUCED BOVINE BLASTOCYSTS BY SUBLETHAL HYDROSTATIC PRESSURE PRETREATMENT

Vitrification of in vitro (IVP)-produced bovine blastocysts is well established, reaching post-warming hatching rates close to 70–80% in vitro. Nevertheless, improvements still are needed regarding realizable pregnancy rates. High hydrostatic pressure (HHP) treatment of fresh boar semen before freezing increased the litter size achieved by insemination of frozen–thawed boar semen (Kuo et al. 2007 6th Int. Conf. Boar Semen Pres, Alliston, ON, Canada, poster #22); HHP treatment-related improvements were also observed in the in vitro cryosurvival of mouse blastocysts (Pribenszky et al. 2004 Reprod. Fertil. Dev. 17, 199–200), bull and boar semen, and pig oocytes, theoretically by the sublethal stress-induced production/stabilization of shock proteins (Pribenszky et al. 2006 Reprod. Fertil. Dev. 18, 162–163; 2007 Reprod. Fertil. Dev. 19, 181–182; and b; Du et al. 2007). The aim of the present study was to improve the post-warming in vitro developmental competence of vitrified bovine IVP blastocysts through the application of HHP as pre-treatment, in order to apply the protocol in later in vivo experiments. Day 7 IVP blastocysts were aspirated in TQC holding medium (AB Technology, Sao Paulo, Brazil) into 0.25-mL straws. Straws were pressure-treated in a custom-made hydrostatic pressure chamber (Cryo-Innovation Ltd., Budapest, Hungary), using water as pressure medium. Six hundred bar pressure was applied for 60 min at 32�C. Immediately after pressure treatment, or following 60- or 120-min incubation, embryos were vitrified and warmed using open pulled straws (OPS) according to the method of Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58). Untreated blastocysts were vitrified as controls. After warming, embryos were cultured in vitro in SOF (Holm et al. 1999 Theriogenology 52, 683–700) for 72 h. Embryos were checked for re-expansion and hatching at 4, 24, 48, and 72 h post-warming. For the experiment, 404 blastocysts were used in 5 replicates. Logistic regression was used for statistical evaluation. All vitrified groups were inferior compared to the non-vitrified control (97%, 97%, 98, and 100% expansion; 0%, 23%, 72 and 91% hatching at 4, 24, 48, and 72 h, respectively). HHP treatment had a significant effect (P &gt; 0.05) on the post-warming developmental competence of vitrified blastocysts. HHP treatment followed by 60 min of equilibration proved to be superior among all treatment groups regarding both re-expansion and hatching rates and the speed of resumption of normal in vitro development (HHP treatment followed by a 60-min equilibration time before vitrification/warming: re-expansion rates: 88%, 89%, 85, and 90%; hatching rates: 0%, 22%, 51, and 73% v. non-treated vitrified/warmed controls: re-expansion rates: 63%, 69%, 71, and 81%; hatching rates: 0%, 6%, 43, and 63%; at 4, 24, 48, and 72 h post-warm, respectively). In conclusion, hydrostatic pressure pre-treatment significantly improved in vitro survival and hatching rates as well as the speed of resumption of normal in vitro development. Further studies are needed to reveal the molecular-biological implications of the HHP treatments, as well as field trials to test if the in vitro improvements can be confirmed by pregnancy and birth rates. This work was supported by EMBRAPA and a Kozma grant, Hungary.

Effect of macromolecules in solutions for vitrification of mature bovine oocytes

This study was designed to evaluate vitrification procedures for in vitro matured bovine oocytes for efficient blastocyst production after warming, IVF and culture. A second goal was to replace serum as the macromolecular component of the vitrification solution, without compromising efficacy. The first experiment compared two containers, open pulled straws (OPS) versus cryoloops, and two vitrification protocols: short equilibration (H-TCM-199 + 10% EG + 10% DMSO + 20% FCS for 30 s, followed by H-TCM-199 + 20% EG + 20% DMSO + 20% FCS + 0.48 M galactose for 20 s) versus long equilibration (H-TCM-199 + 3% EG + 20% FCS for 10 min, followed by H-TCM-199 + 31% EG + 20% FCS + 1 M galactose for 20 s). Subsequent experiments used only cryoloops and the short equilibration protocol to evaluate the effect of replacing FCS with defined macromolecules (BSA, Ficoll, PVP, and PVA) in vitrification solutions. Cryoloops were superior to OPS for vitrification of oocytes as determined by blastocyst production ( P < 0.05). The short and long vitrification protocols gave similar results. The presence of macromolecules in vitrification solutions for bovine oocytes was necessary for acceptable post-warming developmental capacity; 20% FCS, 1% and 2% BSA, 6% and 18% Ficoll, 6% and 20% PVP, 1% PVA, and the combinations of 18% Ficoll + 1% BSA, and 6% PVP + 1% BSA provided similar protection during vitrification of oocytes; development ranged from 14.8% to 23.0% blastocysts/oocyte, which was not different ( P > 0.05) from non-vitrified controls (26.9–34.0% blastocysts/oocyte). Too much (6%) and too little (0.3%) BSA, and 0.3% PVA for vitrification resulted in lower blastocyst production ( P < 0.05) relative to unvitrified oocytes.

132 SUCCESSFUL EMBRYO TRANSFER OF IN VIVO-PRODUCED RED DEER (CERVUS ELAPHUS) EMBRYOS AFTER CRYOPRESERVATION BY SLOW FREEZING OR VITRIFICATION

Multiple ovulation and embryo transfer (MOET) programs for red deer (Cervus elaphus) have been established commercially over the last decade, with embryo cryopreservation being a related practice necessary to enhance the use of valuable genetic information. The aim of this work was to establish alternative methods for red deer embryo cryopreservation by using slow freezing with ethylene glycol (SF–EG) and vitrification by open pulled straw (OPS) methods. After surgical flushing of 18 superstimulated donors, 54 transferable embryos were recovered; 28 were transferred fresh to synchronized recipients and the others were cryopreserved by SF–EG (n = 11) or OPS (n = 15), respectively thawed or warmed, and transferred to recipients. Fresh embryos were maintained in Dulbecco's PBS + 20% cow serum (holding medium, HM) until transfer (maximum 3 h after collection). SF–EG cryopreserved embryos were suspended in HM + 1.78 M EG + 0.1 M sucrose + 4 mg mL−1 BSA. After a 10-min equilibration, embryos were loaded individually into 0.25-mL plastic straws and placed into a −7°C methanol bath chamber. After seeding (5 min later), the straws were cooled from −7 to −35°C at a rate of 0.5°C min. Straws were plunged into and stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 s; their contents were drained into HM until transfer. Embryos were vitrified using the OPS method with minor modifications. They were first incubated in HM + 1.78 M EG + 1.3 M DMSO for 3 min and then transferred for 25 s into a vitrification solution of HM + 3.56 M EG + 2.6 M DMSO + 0.5 M sucrose. Each embryo was loaded by touching a 1-µL drop with the straw, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the straws into HM + 0.25 M sucrose for 5 min. Embryos were then transferred into HM + 0.15 M sucrose for 5 min and finally to HM until transfer. Both types of cryopreserved embryos were transferred a few hours after collection, immediately after thawing or warming. Before embryo transfer, the presence of corpus luteum (CL) of recipients was confirmed by laparoscopic examination. Each embryo was surgically transferred into the apical extreme of the uterine horn ipsilateral to the CL of one recipient. Pregnancy was determined by ultrasonography 41 days after embryo transfer. The pregnancy rate between groups was compared with the chi-square test (P &amp;lt; 0.05). No statistical differences were found between groups (Table 1). Our results show that both vitrification and slow freezing methods with EG are suitable to cryopreserve red deer embryos. Table 1. Pregnancy rates in recipient hinds after transfer of fresh, vitrified, or frozen red deer embryos

Stepwise In-straw Dilution and Direct Transfer Using Open Pulled Straws (OPS) in the Mouse: A Potential Model for Field Manipulation of Vitrified Embryos

In the present study, mouse blastocysts were employed to investigate the feasibility and efficiency of stepwise in-straw dilution and direct transfer using the open pulled straw (OPS) method. In experiment I, the effects of various vitrification solutions (VS) on embryo survival were examined. After thawing, the expanded blastocyst rates (97.59 and 95.05%) and hatching rates (80.48 and 78.95%) achieved in the EDFS30 [15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll, and sucrose] and EFS40 [40% EG, Ficoll, and sucrose] groups were no different from those (96.15% and 83.33%) of the control group. However, the rates in the EFS30 [30% EG, Ficoll, and sucrose] (87.80 and 55.43%) and EDFS40 [20% EG, 20% DMSO, Ficoll, and sucrose] (95.69 and 70.97%) groups were significantly lower than those (96.15 and 83.33%) of the control group (P<0.05). In the experiment II, the effects of the volume of VS in the OPS on the survival of embryos after in-straw thawing were investigated. When the length of the VS in the column was less than 1 cm, the in vitro viability of embryos thawed by stepwise in-straw dilution was no different among the experimental and control groups. The embryos could be successfully thawed by immersing the OPS in 0.5 M sucrose for 3 min and then 0.25 M sucrose for 2 min. In experiment III, the effect of immersion time of the OPS in diluent (PBS) on the viability of vitrified embryos was investigated. After in-straw thawing, OPSs were immersed immediately in 1 ml PBS for 0 to 30 min. When the immersion time of the OPSs in PBS was less than 12 min, in vitro development of the in-straw thawed embryos was no different from that of the controls. In experiment IV, in-straw thawed blastocysts were directly transferred to pseudopregnant mice to examine their in vivo developmental viability. The pregnancy (91.67%) and birth rates (42.42%) of embryos in-straw thawed and directly transferred were no different from those of the unvitrified controls (90.90 and 40%) and embryos thawed by the conventional method (84.61 and 46.94%). These results demonstrate that mouse embryos vitrified with OPS could be successfully thawed by stepwise in-straw dilution and transferred directly to a recipient and that this method might be a model for field manipulation of vitrified embryos in farm animals.

The effect of vitrification in open pulled straws on pregnancy rates after transfer of in vivoproduced bovine embryos

The aim of this study was to compare pregnancy rates after transfer of in vivo produced embryos cryopreserved using open pulled straw (OPS) vitrification (Group V) or conventional freezing method as a control (Group C). Bovine embryos (Day&lt;sub&gt;6.5&amp;ndash;7.5&lt;/sub&gt;) collected from superovulated cows were classified according to developmental stages and morphological qualities (Grade 1 and 2) before cryopreservation and they were transferred to synchronized heifers after thawing. Pregnancy rates after transfer of morulae, early blastocysts and expanded blastocysts in Group V compared to Group C (54.5%, 12/22 vs. 56.0%, 14/25; 53.3%, 16/30 vs. 58.1%, 18/31 and 57.7%, 15/26 vs. 48.3%, 14/29) were not different (P &amp;gt; 0.05). Likewise, pregnancy rates after transfer of embryos of Grade 1 and 2 in Group V compared to Group C (55.1%, 43/78 vs. 54.1%, 46/85 and 36.4%, 12/33 vs. 32.9%, 23/70, respectively) were not different (P &amp;gt; 0.05). The study demonstrated similar viability of embryos which were frozen by vitrification or conventional method irrespective of their quality and developmental stage after transfer into recipients.

Improved survival of vitrified porcine embryos after partial delipation through chemically stimulated lipolysis and inhibition of apoptosis

Mechanical removal of intracellular lipids has been the most effective approach to increase the cryosurvival of porcine embryos. In this experiment, we tested the hypotheses that the cryosurvival of porcine embryos can be improved after partial delipation through chemically stimulated lipolysis and that the survival can be further improved by inhibition of apoptosis. Porcine embryos were produced in vitro using sow oocytes. On Day 5 of embryonic development, embryos were cultured in the presence of 10 μM forskolin for 24 h. On Day 6 blastocysts were vitrified using an open pulled straw (OPS) method and warmed blastocysts were cultured 18 h for them to recover. A caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was used at 20 μM during vitrification and subsequent culture to inhibit apoptosis. A 2 × 2 × 2 factorial design experiment was conducted to examine the effect of chemical delipation, vitrification and apoptosis inhibition. We also measured the lipolytic activity of porcine embryos cultured with or without forskolin. Chemical delipation increased the cryosurvival of porcine embryos compared to the controls (71.2 ± 2.8% versus 37.1 ± 5.1%). Apoptosis inhibition increased the ability of blastocysts to fully recover (23.8 ± 3.1% versus 14.6 ± 4.3%). However, there was no interaction between chemical delipation and apoptosis inhibition. Lipolytic agent treatment increased the lipolytic activity of porcine blastocysts. In conclusion, cryosurvival of porcine embryos was improved by partial delipation through chemical stimulation of lipolysis or apoptosis inhibition.

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

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

Improved development by Taxol pretreatment after vitrification of in vitro matured porcine oocytes

This study was designed to examine the effect of Taxol pretreatment on vitrification of porcine oocytes matured in vitro by an open pulled straw (OPS) method. In the first experiment, the effect of Taxol pretreatment and fluorescein diacetate (FDA) staining on parthenogenetic development of oocytes was evaluated. In the second experiment, viability, microtubule organization and embryo development of oocytes were assessed after oocytes were exposed to vitrification/warming solutions or after vitrification with or without Taxol pretreatment. The results showed that Taxol pretreatment and/or FDA staining did not negatively influence the oocyte's developmental competence after parthenogenetic activation. After being exposed to vitrification/warming solutions, the survival rate (83.3%) of the oocytes was significantly (P < 0.05) reduced as compared with that in the control (100%). Vitrification/warming procedures further reduced the survival rates of oocytes regardless of oocytes being treated with (62.1%) or without (53.8%) Taxol. The proportions of oocytes with normal spindle configuration were significantly reduced after the oocytes were exposed to vitrification/warming solutions (38.5%) or after vitrification with (10.3%) or without (4.1%) Taxol pretreatment as compared with that in control (76.8%). The rates of two-cell-stage (5.6-53.2%) embryos at 48 h and blastocysts (0-3.8%) at 144 h after activation were significantly reduced after exposure to vitrification/warming solutions or after vitrification as compared with control (90.9% and 26.6% respectively). However, the proportion of vitrified oocytes developed to two-cell stage was significantly higher when oocytes were pretreated with (24.3%) than without (5.6%) Taxol. These results indicate that pretreatment of oocytes with Taxol before vitrification helps to reduce the damage induced by vitrification and is a potential way to improve the development of vitrified porcine oocytes.