Open Pulled Straw Method Research Articles

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.

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.

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.

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

Ultrastructure and Development of Vitrified/Warmed Bovine Oocytes Matured with 9-cis Retinoic Acid

In this work we analyze the effects of vitrification on the ultrastructure and developmental ability of bovine oocytes matured in the presence of 9-cis-retinoic acid (RA). Bovine cumulus oocyte complexes (COCs) were matured in a basic medium containing 10% fetal calf serum (FCS), 9-cis-RA or polyvinyl alcohol (PVA). Mature oocytes were vitrified using the Open Pulled Straw method with minor modifications. A group of fresh and vitrified/warmed COCs was fixed for ultrastructural analysis, while the remaining oocytes were fertilized in vitro and cultured. Vitrification of mature oocytes produced high rates of degeneration regardless of the in vitro maturation protocol and low number of cleaved embryos. Development rates up to day 8 were similar between groups undergoing vitrification, which were lower than fresh controls. Morphologic alterations were observed in all groups of vitrified oocytes, although in vitro maturation in the presence of 9-cis-RA combined with vitrification led to higher ultrastructural damage. More research is needed to explain the low survival rates of the bovine oocyte after vitrification and warming.

Numerical investigations of transient heat transfer characteristics and vitrification tendencies in ultra-fast cell cooling processes

During freezing, cells are often damaged directly or indirectly by ice formation. Vitrification is an alternative approach to cryopreservation that avoids ice formation. The common method to achieve vitrification is to use relatively high concentrations of cryoprotectant agents (CPA) in combination with a relatively slow cooling rate. However, high concentrations of CPAs have potentially damaging toxic and/or osmotic effects on cells. Therefore, establishing methods to achieve vitrification with lower concentrations of CPAs through ultra-fast cooling rates would be advantageous in these aspects. These ultra-fast cooling rates can be realized by a cooling system with an ultra-high heat transfer coefficient ( h) between the sample and coolant. The oscillating motion heat pipe (OHP), a novel cooling device utilizing the pressure change to excite the oscillation motion of the liquid plugs and vapor bubbles, can significantly increase h and may fulfill this aim. The current investigation was designed to numerically study the effects of different values of h on the transient heat transfer characteristics and vitrification tendencies of the cell suspension during the cooling processes in an ultra-thin straw (100 μm in diameter). The transient temperature distribution, the cooling rate and the volume ratio ( x) of the ice quantity to the maximum crystallizable ice of the suspension were calculated. From these numerical results, it is concluded that the ultra-high h (>10 4 W/m 2 K) obtained by OHPs could facilitate vitrification by efficiently decreasing x as well as the time to pass through the dangerous temperature region where the maximum ice formation happens. For comparison, OHPs can decrease both of the parameters to less than 20% of those from the widely used open pulled straw methods. Therefore, the OHP method will be a promising approach to improving vitrification tendencies of CPA solutions and could also decrease the required concentration of CPAs for vitrification, both of which are of great importance for the successful cryopreservation of cells by vitrification.

91 PRELIMINARY RESULTS OF SURGICAL EQUINE EMBRYO TRANSFER AFTER OPEN PULLED STRAW VITRIFICATION

In equine species, embryo cryopreservation is not as widely developed as in some other species. Slow freezing has been applied to equine embryos but with relatively low success rates. This higher sensitivity to conventional freezing procedures may be explained by the presence of a capsule surrounding the equine embryo that may impair cryoprotectant penetration. Recently, good in vitro embryo survival rate was obtained after open pulled straw (OPS) vitrification (Moussa et al. 2005 Theriogenology 64, 1619–1632). The aim of the present study was to evaluate in vivo survival of vitrified embryos five days after surgical transfer into Welsh pony mares. Morulae (M), early blastocysts (EB), and blastocysts (B) ranging from 140 to 320 μm in diameter were collected (n = 20) in a Ringer lactate solution on Day 6.75 after ovulation. Before vitrification, embryos were assessed morphologically and their size was measured (McKinnon and Squires 1988 J. Am. Vet. Med. Assoc. 192, 401–406). Then, embryos were vitrified using the OPS method described by Berthelot et al. (2001 Reprod. Nutr. Dev. 41, 267–272). Briefly, embryos were washed twice in HEMES-TCM-199 + 20% newborn calf serum (NBCS) for 1 min, equilibrated in HEPES-TCM-199 + 20% NBCS with 7.5% dimethyl sulfoxide (DMSO) + 7.5% ethylene glycol (EG) for 3 min, and then with 18% DMSO + 18% EG + 0.4 M sucrose for 45 s. One embryo was then loaded per straw. For transfer, four straws were quickly thawed (5 s in air) and the narrow end of the straw containing the embryo was immersed in HEPES-TCM-199 + 20% NBCS + PBS + 0.2 M sucrose. Five to 8 min after thawing, four embryos were surgically transferred into the cranial portion of the uterine horn in each of five pony mare recipients. Five days after transfer, embryos recovered by transcervical flushing of the uterus were classified as viable if morphology was normal, no dark inner cells were present, the capsule was intact, and the diameter was at least 1000 μm. The results are shown in the table. One recipient of vitrified embryos had an endometritis and no embryo was recovered. From the four other recipients, nine embryos were recovered out of 16 (56%) transferred, seven of which were viable (44%). The results of the present preliminary study demonstrating survival of equine embryos transferred after OPS vitrification is very encouraging. However, the results should be confirmed by birth of foals after transfer of OPS-vitrified embryos to recipients. Table

In vitro comparisons of two cryopreservation techniques for equine embryos: Slow-cooling and open pulled straw (OPS) vitrification

Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps with decreasing glycerol and sucrose concentrations. Twenty embryos were vitrified using the OPS method. Embryos were exposed to 7.5% dimethyl-sulfoxide (DMSO) + 7.5% ethylene glycol (EG) for 3 min and in 18% DMSO + 18% EG + 0.4 M sucrose for 1 min, loaded in OPS and plunged into liquid nitrogen. After warming, embryos were placed in decreasing sucrose concentrations. All embryos were cultured in synthetic oviduct fluid (SOF) medium for 3 h and evaluated using 4′,6-diamidino-2-phenylindole (DAPI) staining. The percentage of cells entering in S-phase (%SC) was evaluated by incorporation of BrdU. No significant differences were observed for mean diameter, morphological grade and percentage of degenerate embryos after 3 h of culture for slow-cooling and OPS methods. The percentage of dead cells per embryo was similar for the two procedures (42 ± 6 versus 46 ± 9). The percentage of cells entering in S-phase did not differ significantly between the two procedures (27 ± 5 versus 26 ± 6). OPS vitrification may be as efficient as slow-cooling for the cryopreservation of equine embryos. However, these results should be confirmed by the transfer of OPS vitrified embryos to recipient mares.

Bovine oocytes vitrified by the open pulled straw method and used for somatic cell cloning supported development to term

The objective of the present study was to determine if oocytes vitrified by the open pulled straw (OPS) method could subsequently be used to produce somatic cell cloned cattle. Post-thaw survival rates were 77.0, 79.1, 97.2 and 97.5% for oocytes vitrified with EDFS30 (15% ethylene glycol, 15% dimethyl sulfoxide, ficoll and sucrose), EDFS40 (20% ethylene glycol, 20% dimethyl sulfoxide, ficoll and sucrose), EDFSF30 (15% ethylene glycol, 15% dimethyl sulfoxide, ficoll, sucrose and FBS) and EDFSF40 (20% ethylene glycol, 20% dimethyl sulfoxide, ficoll, sucrose and FBS), respectively. The parthenogenetic blastocyst rates of the vitrified–thawed oocytes activated with 5 μM of the calcium ionophore A23187 for 5 min and 2 μM of 6-dimethylaminopurin (6-DMAP) for 4 h ranged from 10.3 to 23.0%, with the highest group not significantly differing from that of the controls (33.2%). In total, 722 vitrified–thawed oocytes were used as recipients for nuclear transfer, of which 343 fused (47.6%). Fifty-six (16.3%) of the reconstructed embryos reached the blastocyst stage after 7 d of in vitro culture. Twenty-four blastocysts derived from vitrified–thawed oocytes were transferred to six Luxi yellow cattle recipients. Two recipients (33%) were diagnosed pregnant; one aborted 97 d after transfer, whereas the other delivered a cloned calf after 263 d. As a control, 28 synchronous Luxi yellow cattle recipients each received a single blastocyst produced using a fresh oocyte as a nuclear recipient; 10 recipients were diagnosed pregnant, of which 6 (21.4% of the original 28) delivered cloned calves. In conclusion, bovine oocytes vitrified by the OPS method and subsequently thawed supported development (to term) of somatic cell cloned embryos.

Beneficial effects of serum supplementation during in vitro production of porcine embryos on their ability to survive cryopreservation by open pulled straw vitrification

The ability of porcine blastocysts produced in vitro, in the presence or absence of serum, to survive cryopreservation was investigated in this experiment. Porcine oocytes were matured, fertilized and cultured in vitro using serum-free culture systems. Starting at Day 4 of in vitro embryo culture (Day 0 = fertilization), the culture medium was supplemented with 10% fetal bovine serum (FBS). Embryos were cultured under these conditions until Day 6. Embryos cultured with only BSA supplementation served as serum-free controls. Day 6 blastocysts and expanded blastocysts of excellent quality were vitrified using the open pulled straw method. After warming, blastocysts were cultured in the presence of 10% FBS for an additional 18 h to recover. Portions of blastocysts from both groups, without cryopreservation, were also cultured under the same conditions to serve as non-vitrified controls. To further investigate the influence of FBS on the quality of embryos produced, the total cell numbers in Day 6 blastocysts from both groups were compared. In addition, the ratio of viable to total cells in fully recovered blastocysts at each group was examined. Blastocysts produced in the presence of FBS had an increased ability to survive cryopreservation and also had a higher cell number compared to those produced in serum-free systems ( P < 0.05). The fully recovered blastocysts had a normal viable to total cell ratio, compared to non-vitrified controls. Overall, this experiment supports the hypothesis that serum supplementation during in vitro production of porcine embryos is beneficial to the ability of a blastocyst to survive cryopreservation.

Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability

The nuclear stage at which oocytes are cryopreserved influences further development ability and cryopreservation affects ultrastructure of both cumulus cells and the oocyte. In this work, we analyze the effects of vitrification at different nuclear and cytoplasmic maturation stages on the oocyte ultrastructure and developmental ability. Culture in TCM199 + PVA with roscovitine 25 M during 24 h led to meiotic arrest (MA) in cumulus-oocyte complexes (COCs), while permissive in vitro maturation (IVM) was performed in TCM199, 10% FCS, FSH-LH and 17β-estradiol for 24 h. Oocytes were vitrified using the open pulled straw method (OPS) with minor modifications. Fresh and vitrified/warmed COCs were fixed as immature, after IVM, after meiotic arrest (MA) and after MA + IVM. Vitrification combined with MA followed by IVM produced the highest rates of degeneration, regardless of the vitrification time. As a consequence, lower proportions of embryos cleaved in these groups, although differences were eliminated at the five–eight cell stage. Development rates up to day 8 were similar in all experimental groups, being significantly lower than those in fresh controls. Only oocytes vitrified after IVM were able to give blastociysts. The morphological alterations observed can be responsible for compromised development. More research is needed to explain the low survival rates of the bovine oocyte after vitrification and warming.

82 IN VITRO SURVIVAL OF PORCINE BLASTOCYSTS VITRIFIED USING THE CRYOLOGIC VITRIFICATION METHOD

Porcine embryo cryopreservation is an important technology for the storage and transport of valuable genetic material. With many of the current vitrification and storage systems, such as the open pulled straws and microdrops, there is direct contact between the medium containing the embryos and the liquid nitrogen. This represents a possible contamination risk. One system with which there is no direct contact between the embryos and liquid nitrogen during the vitrification process is the Cryologic Vitrification System (CVM; Lindemans et al. Reprod. Fertil. Dev. 16, 174) which uses solid surface vitrification. Microdrops of vitrification medium containing the embryos are placed in contact with a metal block that has been precooled by partial submersion in liquid nitrogen, resulting in very rapid cooling rates. Blastocysts were collected surgically on day 5 of pregnancy from mature sows, and the embryos were randomly divided into two groups; each group was then vitrified and warmed with either of two previously published protocols except that the CVM replaced the open pulled straws plunged into liquid nitrogen in both protocols. The first method (OPS/CVM) was based on the open pulled straw method (Cuello et al. Theriogenology 61, 843-850), and used DMSO and ethylene glycol as cryoprotectants and TCM-199 as the basic medium. The second method (EG/CVM) used HEPES-buffered NCSU23 as the basic medium; the blastocysts were centrifuged prior to vitrification in ethylene glycol and polyvinylpyrrolidone (PVP) and the zona pellucida was removed immediately after warming (Cameron et al. Theriogenology 61, 1533-1543). Embryos were then cultured in NCSU23 +10% fetal bovine serum for 48 h at 38.5�C in an humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Embryos that had reformed the blastocoel and continued to expand were considered to have survived. These were stained with Hoechst 33342 and the nuclei counted using fluorescence microscopy. There was no difference between the OPS/CVM or EG/CVM methods in either the survival rates (27/29; 93%, and 24/27; 89%, respectively) or the number of cells (mean � SEM; 109 � 6 and 112 � 6, respectively). The survival rates are comparable to previously published rates using these two methods and open pulled straws. These data suggest that the CVM can successfully replace the open pulled straws in these two protocols. However, transfer of vitrified and warmed embryos into recipients would be needed to confirm the viability of the surviving embryos.

Vitrification of calf oocytes: Effects of maturation stage and prematuration treatment on the nuclear and cytoskeletal components of oocytes and their subsequent development

This study was designed to establish the effects of the meiotic stage of bovine oocytes and of a prematuration treatment with roscovitine (ROS) on their resistance to cryopreservation. Oocytes from prepubertal calves at the stages of germinal vesicle breakdown (GVBD) or at metaphase II (MII) were vitrified by the open pulled straw (OPS) method. In another experiment, oocytes were kept under meiotic arrest with 50 microM ROS for 24 hr and vitrified at the GVBD stage. After warming, some oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. Significantly lower cleavage rates were obtained for the vitrified GVBD and MII oocytes (9.9% and 12.6%, respectively) compared to control oocytes (73.9%). Significantly worse results in terms of cleavage rates were obtained when GVBD calf oocytes were exposed to cryoprotectants (CPAs: ethylene glycol plus dimethyl sulfoxide, DMSO) (13.1%) or vitrified (1.6%) after a prematuration treatment with ROS, when compared to untreated control oocytes (68.7%) or ROS-control oocytes (56.6%). None of the vitrification procedures yielded blastocysts, irrespective of the initial meiotic stage or previous prematuration treatment. Compared to the control oocytes, significantly fewer oocytes exhibited normal spindle configuration after being exposed to CPAs or after vitrification of either GVBD or MII calf oocytes. These results indicate that the vitrification protocol has a deleterious effect on the meiotic spindle organization of calf oocytes cryopreserved at both the GVBD and MII stage, which impairs the capacity for further development of the embryos derived from these vitrified oocytes. Prematuration treatment with ROS has no beneficial effect on the outcome of vitrification by the OPS method.

82COMPARISON OF THE SURVIVAL OF IN VITRO-DERIVED PORCINE OOCYTES AND EMBRYOS VITRIFIED BY OPEN PULLED STRAW, ELECTRON MICROSCOPE GRID, AND NYLON LOOP SYSTEM

Various sample containers have been developed for ultra-rapid vitrification of oocytes and embryos to minimize chilling and other injuries. In this study, the survival rates of in vitro-derived porcine oocytes were compared after cryopreservation using open pulled straw (OPS), electron microscope grid (EMG), and nylon loop system (NLS). In addition, the post-thaw survival of porcine morulae and blastocysts was assessed after vitrification by the OPS method. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al., 1994 Theriogenology 41, 1425–1433). Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5mM sodium pyruvate, 0.5mM sodium lactate and 4mgmL−1 bovine serum albumin for 2 days at 39°C; 10% fetal bovine serum was added to the culture medium thereafter. Oocytes and embryos were treated with 7.5μgmL−1 cytochalasin B for 30min, centrifuged at 13,000g for 13min and then exposed sequentially to an ethylene glycol (EG) vitrification solution (0M for 1min, 2M for 5min and 8M plus 7% polyvinylpyrrolidone for 1min). Oocytes (n=100per treatment group) were placed in or on sample containers and plunged into liquid nitrogen. Porcine morulae and blastocysts (n=137) were similarly treated, aspirated into OPSs, and plunged into liquid nitrogen. Oocytes and embryos were thawed rapidly by transferring the sample container into 1M EG for 2min, and then the embryos were serially diluted by transfer into 0.5M for 2min, and medium for 5min. Post-thaw survival of vitrified oocytes was assessed by development after IVF and culture to morulae or blastocysts. Post-thaw survival of vitrified morulae and blastocysts was assessed by their ability to continue development to, respectively, blastocysts and expanded blastocysts. After oocytes were thawed, fertilized and cultured in vitro, the rates of development to the morula- or blastocyst-stage were 8%, 0% and 6% for, respectively, the OPS, EMG and NLS groups. The rate of development of non-cryopreserved control oocytes was 38% (38/100). Although significantly fewer oocytes developed after vitrification compared to the control (P&amp;lt;0.05), OPS and NLS containers were superior to EMG containers (P&amp;lt;0.05). Morula- or blastocyst-stage embryos vitrified using OPS yielded a significantly higher rate of survival than did oocytes (48%, 66/137; P&amp;lt;0.05). This study indicates that very few in vitro-matured porcine oocytes survive vitrification using OPS, EMG and NLS methods. Considerably higher rates of survival were obtained by in vitro-produced porcine morulae and blastocysts following vitrification by the OPS method. Additional research is needed to identify and develop methods to overcome the factors limiting the cryopreservation of porcine oocytes for practical uses. Supported by KOSEF (R05-2002-000-00388-0) grant.

105VITRIFICATION OF BOVINE EMBRYOS USING THE CLV METHOD

Vitrification has become the preferred method for cryopreserving in vitro-produced bovine embryos (IVP). Here we introduce a technique for vitrification developed at CryoLogic (the CLV Method), in conjunction with a study comparing the post-thaw viability of IVP embryos frozen by the widely used open pulled straw method (OPS—Vajta et al., 1997 Cryo-Letters 18, 191) and the new CLV Method. Vitrification on thin metal surfaces has been explored and demonstrated previously (Le Gal &amp;amp; Massip 1999, Cryobiology 38, 290), and Dinnyes presented a Solid Surface Vitrification (SSV) (Dinnyes et al., 2000, Biol. Reprod. 63, 513). The CLV Method utilizes vitrification on the surface of a solid metal block. This surface has been custom shaped and treated to enhance vitreous formation. The method also includes handling, storage and thawing protocols designed to avoid damage from crystallization of the unstable glass. Briefly, the block is precooled in LN2 to −196°C. Up to 10 embryos are collected into a droplet of medium (3μL), on the end of a fibre carrier attached to a handle. The droplet is presented to the vitrification surface, where it is converted into a glass bead by cooling rates comparable to that of plunging into solid/liquid phase nitrogen (−210°C) (Arav et al., 2001 Theriogenology 55, 313). The glass bead, fibre and handle are transferred quickly into a half-sealed, precooled straw, and the handle seals the open end. A bead is thawed very rapidly by removing the handle, fibre and bead from the straw and transferring the bead into washing medium. COCs collected from bovine ovaries obtained from abattoirs were matured, fertilized and cultured for 6 days (Fry et al., 2003 Theriogenology 59, 446). Embryos reaching the blastocyst or expanding blastocyst stage of development were graded (Grades 1, 2, and 3), equilibrated for 5min in HEPES-199 medium with 20% FCS (HFm), placed in HFm with 10% EG, and 10% DMSO (VS1) for 2min, and then transferred to HFm with 20% EG, 20% DMSO (VS2) for 30s (Vajta). Between 5 and 10 IVP embryos were processed and collected for vitrification, either in an OPS plunged into LN2, or in a 3μL droplet vitrified by the CLV Method. The two sets of specimens were stored in LN2, and later thawed. Both OPS tips and beads were thawed in 0.5mL of HFm with 0.2M sucrose at 39°C. Embryos were maintained at 39°C, examined after 5min for contraction, and again after 6 h for re-expansion. They were then transferred to culture medium, incubated and examined at 24 and 48h to assess hatching. As shown in Table 1, the CLV method appears to be satisfactory for maintaining membrane integrity (expansion) and developmental potential (hatching) for even poorer grade embryos, that might be more sensitive to the stresses of cryopreservation. Table 1 Re-expansion and hatching rates of graded thawed bovine embryos vitrified by OPS or CLV methods

Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP)

The purpose of this study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of in vitro-produced (IVP) bovine blastocysts and to compare the results with post-thaw survival rate of bovine blastocysts frozen in GMP with those frozen in open pulled straw (OPS) that have been previously investigated. The GMP vessel permitted higher freezing and warming rates than the OPS due to the higher heat conductivity of the glass and the lower mass of the solution that contained the embryos. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into either the OPS or GMP vessels and they immersed into LN 2 within 20–25 s. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in a holding medium (HM: D-PBS supplemented with 5% FCS) and then in TCM-199 for 5 min in both cases. They was then cultured in TCM 199 supplemented with 10% FCS for 24 or 48 h. The rate of blastocyst re-expansion was significantly different between OPS (79.6%) and GMP (90.4%) methods. Neither was the hatching rate significantly different among OPS (51.8%), GMP (57.1%) methods and non-vitrified group (67.3%). Only the rate of post-thaw re-expanding of blastocysts loaded in narrow column was significantly higher than that of the wide column (83.3% versus 56.7%) ( P<0.05), although the GMP straw was loaded with three blastocysts per vessel. These results indicated that the GMP vessels provided high survival rates of bovine IVP blastocysts. The location of the embryos loaded into a narrow or wide portion was considered to be a limiting factor to the viability of bovine IVP embryos.