Minimum Volume Cooling Research Articles

A 3D-Printed Large Holding Capacity Device for Minimum Volume Cooling Vitrification of Embryos in Prolific Livestock Species

Although many devices have been developed to reduce sample volume, with an explosion of methods appearing in the literature over the last decade, commercially available devices with simultaneous vitrification of a larger number of embryos are scarce, with the apparent gap for their use in prolific livestock species. In this study, we investigated the effectiveness of a new three-dimensional (3D)-printed device that combines minimum volume cooling vitrification with simultaneous vitrification of a larger number of rabbit embryos. Late morulae/early blastocysts were vitrified with the open Cryoeyelet® device (n = 175; 25 embryos per device), the open Cryotop® device (n = 175; 10 embryos per device), and the traditional closed French mini-straw device (n = 125; 25 embryos per straw) and compared in terms of in vitro development and reproductive performance after transfer to adoptive mothers. Fresh embryos constituted the control group (n = 125). In experiment 1, there was no difference in the development rate to the blastocyst hatching stage between the CryoEyelet® and the other devices. In experiment 2, the CryoEyelet® device showed a higher implantation rate compared with the Cryotop® (6.3% unit of SD, p = 0.87) and French mini-straw® (16.8% unit of SD, p = 1.00) devices. In terms of offspring rate, the CryoEyelet® device was similar to the Cryotop® device but superior to the French straw device. Regarding embryonic and fetal losses, the CryoEyelet® showed lower embryonic losses compared to other vitrification devices. The analysis of bodyweight showed that all devices showed a similar outcomes-a higher birthweight but a lower body weight at puberty than those in the fresh transfer embryos group. In summary, the CryoEyelet® device can be used for the vitrification of many late morulae or early blastocyst stage rabbit embryos per device. Further studies should be performed to evaluate the CryoEyelet® device in other polytocous species for the simultaneous vitrification of a large number of embryos.

Cryodevices developed for minimum volume cooling vitrification of bovine oocytes.

Unfertilized bovine oocytes can be efficiently cryopreserved only when an extremely rapid cooling rate (>20,000°C/min) is applied to oocytes with a very limited amount of surrounding vitrification solution. This protocol is defined as minimum volume cooling (MVC) vitrification. Various types of cryodevices, such as open pulled straw, Cryoloop, and Cryotop, have been developed to accelerate the cooling efficacy. Furthermore, hollow fibers with nano‐scale pores, triangle nylon mesh sheets, and multilayer silk fibroin sheets have been optimized for the loading of large quantities of oocytes and/or the subsequent removal of excess vitrification solution, without requiring skillful operation to transfer individual oocytes using fine capillaries. This article provides an up‐to‐date review of cryodevices suitable for the MVC vitrification of bovine oocytes at the immature (germinal vesicle‐) and mature (metaphase II‐) stages.

Silk fibroin sheet multilayer suitable for vitrification of in vitro-matured bovine oocytes

Minimum volume cooling (MVC) procedure has been successfully applied to vitrify mammalian oocytes, but high skill of capillary pipetting is required to load the oocytes on a cryodevice with a minimal volume (<1 μL) of vitrification solution (VS). Here we report a novel cryodevice for bovine oocyte vitrification, silk fibroin (SF) sheet multilayer, of which spontaneous absorption property can eliminate pipette operation for removal of excess VS. Based on physical stability and scanning electron microscopic observation, the SF sheet prepared from 1.5% (wt/vol) fibroin solution was selected and layered around a polypropylene strip (0.1-mm thickness, 0.7-mm width, 10-mm depth). Ten denuded bovine mature oocytes were loaded onto the SF sheet multilayer with 2–3 μL of the VS, and then cooled rapidly by plunging into liquid nitrogen. Nylon mesh (NM) device with square opening 37-μm length of a side and commercially available Cryotop® (CT) device were used as controls, and the minimization of VS volume was performed by paper towel absorption and capillary aspiration, respectively. In SF, NM and CT groups, post-warming oocyte recovery rates were 99.5, 99.1 and 100%, and the morphological survival rates were 99.7, 94.5 and 99.0%, respectively. Subsequent IVF and 8-days IVC resulted in comparable blastocyst yields among the three groups (25.5, 25.0 and 26.1% in SF, NM and CT groups, respectively). These results suggest that SF sheet multilayer is a useful cryodevice for bovine matured oocytes in MVC vitrification because VS volume surrounding the oocytes can be easily minimized through its absorption property.

Vitrification for bovine embryos with low-quality grade.

This study was conducted to examine the utility of vitrification for bovine embryos with low-quality grade, and simple cryoprotectants dilution method for practitioners. In Experiment 1, survival of frozen embryos was compared with that of vitrified embryos using minimum volume cooling (MVC). Then, vitrified embryos were used to confirm the optimum sucrose concentration in Experiment 2. The survival rates of embryos that had been vitrified following diluted cryoprotectants with the one-step in-straw method were compared with those of fresh control embryos in Experiment 3. Frozen-thawed or vitrified-warmed blastocysts were cultured with TCM-199 supplemented with 100μmol/L beta-mercaptoethanol +5% fetal bovine serum at 38.5°C in an atmosphere of 5% CO2 in air, their survival after 24hr were compared. The development to term of fair quality invivo embryos after vitrification was examined in Experiment 4. Results show that survival rates of frozen-thawed embryos were lower (p<.05) than that of vitrified-warmed ones. When vitrified embryos were warmed in 0.3mol/L sucrose in straws, their survival rate was 100%. The total cell numbers of vitrified-warmed embryos were comparable to those of fresh control embryos. The six calves from 13 vitrified embryos were delivered in Experiment 4. These results indicate that MVC vitrification following one-step cryoprotectants dilution is utilized to preserve low-quality bovine embryos.

Comparison of the microdrop and minimum volume cooling methods for vitrification of porcine &lt;i&gt;in vitro&lt;/i&gt;-produced zygotes and blastocysts after equilibration in low concentrations of cryoprotectant agents

We compared the efficacy of the microdrop and minimum volume cooling (MVC) methods for the vitrification of in vitro-produced porcine zygotes and blastocysts after equilibration in low concentrations of cryoprotectant agents. Zygotes and blastocysts were equilibrated in 2% (v/v) ethylene glycol and 2% (v/v) propylene glycol for 13–15 min. Then, they were vitrified in a medium comprised of 17.5% ethylene glycol, 17.5% propylene glycol, 0.3 M sucrose, and 50 mg/ml polyvinylpyrrolidone either by either dropping them directly into liquid nitrogen (microdrop method) or placing them on Cryotop sheets in a minimum volume of medium and plunging into liquid nitrogen (MVC method). Both zygotes and blastocysts were successfully vitrified. For the vitrification of zygotes, the MVC and microdrop methods were equally effective; however, for blastocyst vitrification, MVC was superior. For both methods, the vitrification of zygotes produced higher-quality embryos than the vitrification of blastocysts.

An efficient and mass reproducible method for vitrifying mouse embryos on a paper in cryotubes

To date, the most successful and popular vitrification method is based on the minimum volume cooling (MVC) concept, in which embryos are vitrified in a very small volume of vitrification solution (VS) and then stored in cryotubes in liquid nitrogen (LN2). Unfortunately, these methods need special devices and may not be suitable for vitrifying a large number of embryos. Theoretically, more embryos in VS on a paper (MVC concept) in cryotubes can be vitrified effectively. Therefore, this study directly vitrifies mouse embryos on a Kimwipes tissue in an 1.8mL cryotube. The ICR 2-celled to blastocyst embryos were used for testing this procedure. In Treatment 1, embryos transferred with 1–2μL of VS into a cryotube. Treatment 2 was similar to Treatment 1 except that the cryotube was filled with LN2. Treatment 3 was identical to Treatment 1 except that a small piece (5mm2) of a sterilized Kimwipes tissue was placed on the top of VS. Treatment 4 was identical to Treatment 3 except for the cryotube being filled with LN2. After each treatment, the cryotubes were capped and transferred to a LN2 tank. After warming, the recovered embryos were cultured in KSOM+AA for 1–3days. There were no differences in the recovery rate, overnight survival rate, blastocyst rate, and birth rate after embryo transfer among all treatment groups. Our results demonstrated an alternative simple, efficient, and mass reproducible method for vitrifying mouse embryos using papers as a vehicle and cryotubes as a container.

130 EFFECT OF HYALURONIC ACID SUPPLEMENTATION ON OVINE EMBRYO DEVELOPMENT IN VITRO AND ON SURVIVAL AFTER VITRIFICATION

Studies have shown that supplementation with hyaluronic acid (HA), a glycosaminoglycan found in mammalian follicular, oviduct, and uterine fluids, improves in vitro development and post-thaw survival of bovine embryos. In this study, we examined the effect of HA supplementation on ovine embryo development and on survival after vitrification using the minimum volume cooling (MVC) cryotop method. Abattoir sourced ovine oocytes were in vitro matured and fertilized as per routine procedures (Walker et al. 1996 Biol. Reprod. 55, 703–708). In Experiment 1 (5 replicates), presumptive zygotes were randomly allocated to IVC medium supplemented with 0.8 mg mL–1 BSA, amino acids, and 0, 0.5, 1.0, 1.5 or 2.0 mg mL–1 HA. Cleavage rates were recorded and blastocyst development evaluated on Day 7 (Day 0 = day of IVF). In Experiment 2 (3 replicates), presumptive zygotes were placed in in vitro culture (IVC) medium with or without 1.0 mg mL–1 HA. Embryos were vitrified using the MVC cryotop method (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172) on either Day 5 (morula–blastocyst stages), Day 6 (compact morula–hatching blastocyst stages), or Day 7 (blastocyst–hatching blastocyst stages). Vitrified embryos were thawed 7 days later and placed into IVC medium. Embryo survival (assessed by blastocoele re-expansion) and hatching rates were recorded on Day 8. Variables were assessed using procedure CATMOD in SAS (SAS Institute Inc., Cary, NC, USA). In Experiment 1, the addition of HA did not affect cleavage or blastocyst formation rates but hatching rates were significantly (P &lt; 0.05) improved at concentrations of 0.5 to 1.5 mg mL–1 (Table 1). In Experiment 2, HA supplementation (1.0 mg mL–1) compared with control medium did not affect cleavage (96.8 and 97.1%, respectively) or blastocyst formation rates (68.6 and 70.2%, respectively). HA significantly (P &lt; 0.05) improved survival after thawing of embryos vitrified on Day 5 (100 v. 85.6%, n = 85 and 90). However no effect was observed when embryos were vitrified on either Day 6 (97.8 v. 97.8%, n = 91 and 92) or Day 7 (96.7 v. 97.9%, n = 92 and 94). Within day, HA supplementation did not affect hatching rate compared with control medium (Day 5, 54.1 v. 53.2%; Day 6, 62.9 v. 65.6%; Day 7, 71.9 v. 62.0%, respectively). These results demonstrate that HA supplementation of IVC medium significantly improves hatching rate of ovine embryos and we speculate that this improvement may correlate with comparable improvements in pregnancy rates after transfer. Hyaluronic acid binds to CD44, a glycoprotein expressed on the surface of preimplantation ovine embryos and shown to play a role on embryo development (Luz et al. 2012 Genet. Mol. Res. 11, 799–809). Hyaluronic acid plays a role in cell migration and we suggest that, in the early blastocyst, it affords an advantage to the trophectoderm cells. Table 1.Effect of hyaluronic acid (HA) supplementation in IVC medium on cleavage, blastocyst, and hatching rates

Hollow Fiber Vitrification: A Novel Method for Vitrifying Multiple Embryos in a Single Device

Current embryo vitrification methods with proven efficacy are based on the minimum volume cooling (MVC) concept by which embryos are vitrified and rewarmed ultrarapidly in a very small amount of cryopreserving solution to ensure the high viability of the embryos. However, these methods are not suitable for simultaneously vitrifying a large number of embryos. Here, we describe a novel vitrification method based on use of a hollow fiber device, which can easily hold as many as 40 mouse or 20 porcine embryos in less than 0.1 μl of solution. Survival rates of up to 100% were obtained for mouse embryos vitrified in the presence of 15% DMSO, 15% ethylene glycol and 0.5 M sucrose using the hollow fiber vitrification (HFV) method, regardless of the developmental stage of the embryos (1-cell, 2-cell, morula or blastocyst; n = 50/group). The HFV method was also proven to be effective for vitrifying porcine in vitro- and in vivo-derived embryos that are known to be highly cryosensitive. For porcine embryos, the blastocyst formation rate of in vitro maturation (IVM)-derived parthenogenetic morulae after vitrification (48/65, 73.8%) did not decrease significantly compared with non-vitrified embryos (59/65, 90.8%). Transfer of 72 in vivo-derived embryos vitrified at the morula/early blastocyst stages to 3 recipients gave rise to 29 (40.3%) piglets. These data demonstrate that the HFV method enables simultaneous vitrification of multiple embryos while still adhering to the MVC concept, and this new method is very effective for cryopreserving embryos of mice and pigs.

In Vitro Development and Postvitrification Survival of Cloned Feline Embryos Derived from Preadipocytes

The aim of the present study was to optimize the conditions for in vitro development and postvitrification survival of somatic cell cloned feline embryos. To determine the effects of cell cycle synchronization of the nuclear donor cells, we cultured preadipocytes under serum starvation or conventional conditions. After two days in serum starvation culture, the proportion of synchronized donor cells at the G0/G1 phase was 91.6%. This was significantly higher than the proportion of non-synchronized cells in the proliferative phase (72.6%, P<0.05). The in vitro development of somatic cell nuclear transfer (SCNT) embryos reconstructed using donor cells treated under serum starvation conditions (normal cleavage rate of 65.7%, 46/70, and blastocyst formation rate of 20.0%, 14/70) was comparable to that of the serum supplemented group (52.5%, 31/59, and 20.3%, 12/59). Use of in vitro or in vivo matured oocytes as recipient cytoplasts equally supported development of the SCNT embryos to the blastocyst stage (11.9%, 5/42, vs. 9.5%, 2/21). SCNT-derived blastocysts were vitrified using the original minimum volume cooling (MVC) or the modified (stepwise) MVC method. Although none (n=10) of the SCNT blastocysts survived following vitrification by the original MVC method, the stepwise MVC method resulted in 100% survival after rewarming (n=11). In conclusion, we demonstrated that feline somatic cell cloned embryos with a high developmental ability can be produced irrespective of cell cycle synchronization of donor cells using either in vivo or in vitro matured oocytes. Furthermore, by utilizing a stepwise vitrification method, we showed that it is possible to cryopreserve cloned feline blastocysts.

Impact of phase transition on the mouse oocyte spindle during vitrification

During vitrification, the glass-like solidification is the phase-transition process from liquid to solid. Phase transition is one of the major factors suspected to affect the physiology of the oocyte, such as the structure of the meiotic spindle. Therefore, it is very important to investigate the systematic and morphological alterations of the metaphase-II spindle and chromosome arrangement during complete course of a vitrification and warming process. B6D2F1 (C57BL/6 X DBA/2) mouse oocytes were cryopreserved by minimum volume cooling (MVC) method of vitrification in a solution with 15% ethylene glycol, 15% dimethylsulphoxide and 0.5 mol/l sucrose. To examine the spindle, oocytes were fixed before, during and after vitrification and were analysed by immunocytochemistry and confocal microscopy. It was shown that spindles in all oocytes could be maintained through the vitrification and warming process, even though they were exposed to extreme temperature and two rounds of phase transition. According to the sequential observations, chromosome alignment was maintained throughout the complete course of vitrification, warming and post-warming stage. The impact of phase transition was barely detectable when the oocyte was exposed to the vitrification and warming process. The oocyte spindle was able to recover immediately after warming.

85 VITRIFICATION OF IN VITRO-MATURED PORCINE OOCYTES AT THE METAPHASE-II STAGE

Cryopreservation of mammalian metaphase-II (M-II) oocytes is still impractical compared to that of early stage embryos. In this study we examined the effects of delipation and mitotic spindle stabilization in order to improve the post-vitrification survival rate of in vitro-matured (IVM) porcine oocytes at the M-II stage. Cumulus–oocyte complexes that had been collected from slaughterhouse ovaries were matured in vitro in NCSU23 supplemented with 0.6 mm cysteine, 10 ng mL–1 epidermal growth factor (EGF), 10% porcine follcular fluid (PFF), and 10 IU mL–1 eCG and hCG. The denuded M-II oocytes were vitrified in the presence of 30% ethylene glycol and 0.5 m sucrose using the minimum volume cooling (MVC) method with a MVC plate (Cryotop�; Kitazato Supply, Tokyo, Japan). Vitrified embryos were rewarmed by immersing the MVC plate directly into rewarming solution containing 1 m sucrose and 20% calf serum at 39�C for 1 min, followed by stepwise dilution of the cryoprotectants. We compared the effects of previtrification treatments, namely, (1) delipation, (2) mitotic spindle stabilization, (3) delipation + mitotic spindle stabilization, and (4) no treatment. For delipation, we used a noninvasive method (Esaki et al. 2004 Biol. Reprod. 71, 432–437) that we had published previously with slight modification. The embryos were treated with 4% trypsin at 38�C for approximately two min to expand the zona pellucida, and then centrifuged (12 000g, 38�C 23 min) with 7.5 µg mL–1 cytochalasin B to polarize cytoplasmic lipid droplets within the perivitelline space. For mitotic spindle stabilization, M-II oocytes were vitrified in the presence of 1 µm paclitaxel. After the oocytes were rewarmed, electrical activation of the oocytes (150 V mm–1, 100 µs, one time) was carried out to induce parthenogenesis. These parthenogenetic embryos were cultured in PZM-5 for 7 days, and the number of vitrified embryos that developed into blastocysts with respect to each treatment was determined. The blastcyst formation rate and mean cell numbers of the blastcysts were compared among the treatment groups (chi-square test, Tukey's test). Of the 50 M-II oocytes that had been vitrified without pretreatment, only one oocyte (2.0%) developed into a blastocyst with 20 cells. By contrast, the number of vitrified embryos that developed into blastocysts was significantly high when they were delipated prior to vitrification (37.8%, 14/37, 64.0 � 9.6; P &lt; 0.01). Mitotic spindle stabilization also improved the survival rate of vitrified oocytes (18.6%, 21/113, 56.7 � 9.6; P &lt; 0.01). The combination of delipation and mitotic spindle stabilization produced the highest number of vitrified oocytes that developed into blastocysts (43.8%, 35/80, 69.4 � 6.4), although the difference between the combination group and the delipation group was not significant. These results indicate that blastocysts can be produced very efficiently from IVM porcine oocytes that have been vitrified at the M-II stage using both noninvasive delipation and mitotic spindle stabilization procedures.

84 PRODUCTION OF CLONED PIGLETS FROM NUCLEAR TRANSFER EMBRYOS AFTER VITRIFICATION

Cryopreservation of cloned embryos is expected to be beneficial in improving the efficiency of somatic cell cloning in pigs. We have already demonstrated that normal piglets can be produced from in vitro-matured and fertilized (IVM/IVF) embryos vitrified at an early cleavage stage after delipation (Nagashima et al. 2007 Biol. Reprod. 76, 900–905). In this study we utilized this technique in an attempt to produce piglets from cloned embryos reconstructed with IVM oocytes. Nuclear transfer (NT) embryos were reconstructed using oocytes matured in vitro in NCSU23 and preadipocytes as nuclear donors. The embryos were cultured in PZM-5 for approximately 98 h, and those that had developed to the morula stage were delipated using a noninvasive method described previously (Esaki et al. 2004 Biol. Reprod. 71, 432–437). The embryos were treated with 4% trypsin at 38�C for 1 to 4 min to induce a slight swelling of the zona pellucida, and then centrifuged (12 000g, 38�C, 23 min) with 7.5 µg mL–1 cytochalasin B to polarize cytoplasmic lipid droplets within the perivitelline space. The embryos were cultured for 1 h and vitrified by the minimum volume cooling (MVC) method using a MVC plate (Cryotop�; Kitasato Supply Co., Tokyo, Japan) in the presence of 15% ethylene glycol, 15% DMSO, and 0.5 m sucrose as cryoprotectants. Vitrified embryos were rewarmed by immersing the MVC plate diretly into rewarming solution containing 1 m sucrose and 20% calf serum at 38�C for 1 min, followed by stepwise dilution of the cryoprotectants. The rewarmed embryos were cultured for 2 days to the blastocyst stage, and then treated with 0.5% pronase to remove the zona pellucida before transfer to the uterine horn of recipients. A total of 103 vitrified blastocysts were transferred to 2 recipient gilts. Both gilts became pregnant and farrowed 2 and 4 piglets, respectively (6/103, 5.8%). These results demonstrate that cloned piglets can be produced from NT embryos that have been cryopreserved at the morula stage using noninvasive delipation and vitrification procedures. This study was supported by PROBRAIN.

126 CRYOPRESERVATION OF PORCINE EMBRYOS DERIVED FROM IVM OOCYTES

We have reported that a combination of delipation (removal of cytoplasmic lipid droplets from blastomeres) and vitrification by means of the minimum-volume cooling (MVC) method successfully cryopreserves porcine in vitro-matured/fertilized (IVM/IVF) embryos, and that normal piglets are produced from these embryos (Hiruma et al. 2006 Reprod. Fertil. Dev. 18, 157). We have also reported that IVM-derived embryos that undergo noninvasive delipation (i.e. micromanipulation is not required) and vitrification develop into blastocysts at a high rate (Esaki et al. 2004 Biol. Reprod. 71, 432–437). In this study, we examined whether fetuses can be produced from the IVM-derived embryos that have been delipated noninvasively and vitrified. Cumulus–oocyte complexes that had been collected from slaughterhouse ovaries were in vitro-matured in NCSU23 medium. The IVM oocytes were activated to produce parthenogenetic embryos. We used the embryos at the 4- to 8-cell (67 h after activation) and morula (98 h) stages in the following experiments. Embryos were treated with 4% trypsin (in PBS) at 38�C for 1 to 4 min to expand the zona pellucida. Next, the embryos were centrifuged (12 000g, 38�C, 23 min) in TL-HEPES-PVP containing 7.5 �g mL-1 cytochalasin B to polarize cytoplasmic lipid droplets within the perivitelline space. These embryos were cultured for 1 to 3 h and then vitrified. The post-thaw viability of the embryos was assessed based on their ability to develop into blastocysts and fetuses (21 to 23 days old). The embryos were vitrified using the MVC method with 15% ethylene glycol, 15% DMSO, and 0.5 M sucrose as cryoprotective agents. PZM-5 was used for culturing the embryos. In embryo transfer experiments, after thawing, the embryos were cultured for 36 or 72 h until they developed into morulae or 4- to 8-cell blastocysts, respectively; they were then treated with 0.5% pronase to remove the zona pellucida, and transferred to the uterine horns of estrus-synchronized recipients 6 days after onset of estrus. The proportion of vitrified embryos that developed into blastocysts and the mean cell number of the blastocysts were similar to those of non-vitrified control embryos, irrespective of the embryonic stage (4- to 8-cell stage: 42.1%, 22/51, 63.0 � 7.8 vs. 64.7%, 22/34, 74.2 � 7.1, respectively; morula stage: 77.6%, 38/49, 69.6 � 7.2 vs. 83.3%, 45/54, 66.2 � 5.9, respectively). Seventeen embryos that had been vitrified at the 4- to 8-cell stage gave rise to 3 fetuses after transfer into one recipient (17.6%). Fifty-three embryos that had been vitrified at the morula stage were transferred into 3 recipients. All recipients became pregnant and produced a total of 17 fetuses (32.1%). These results suggest that porcine IVM-derived embryos that have been cryopreserved by the combination of noninvasive delipation and vitrification by the MVC method are highly viable.

Update on Clinical Efficiency of the Vitrification Method for Human Oocytes in an In Vitro Fertilization Program

Oocyte cryopreservation can be a useful tool in IVF/ICSI-ET cycles of human ART. Time allowance between oocyte retrieval and insemination by cryopreservation gives chances for certain female cancer patients to avoid iatrogenic sterility from chemo/radiotherapy. For patients who desire to delay their child bearing, long term preservation provides an opportunity to have the babies of their owns. Present experiments were conducted to evaluate the clinical efficiency of the minimum volume cooling (MVC) vitrification method for human oocytes in IVF cycles.

98 SUCCESSFUL PREGNANCIES FOLLOWING TRANSFER OF VITRIFIED PORCINE EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES

To date only in vivo-produced embryos have successfully produced live piglets after cryopreservation. In this study, we aimed to produce piglets from vitrified embryos derived from in vitro matured (IVM) oocytes. Cumulus-oocyte complexes collected from ovaries obtained at a local slaughterhouse were matured for 44 to 45 h in NCSU23 MEDIUM supplemented with 0.6 mM cysteine, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 75 �g/mL potassium penicillin G, 50 �g/mL streptomycin sulfate, and 10 IU/mL eCG/ hCG. These IVM oocytes were either activated for parthenogenesis or in vitro-fertilized (IVF). For IVF, oocytes were incubated with 5 � 106/mL of cryopreserved epididymal sperm in PGM-tac medium (Yoshioka et al. 2003 Biol. Reprod. 69, 2092-2099) for 20 h. Embryos were treated for removal of cytoplasmic lipid droplets (delipation; Nagashima et al. 1995 Nature 374, 416) at the 4- to 8-cell stages, around 50 to 54 h after activation or insemination. After culture in NCSU23 for 15 h, they were vitrified by the minimum volume cooling (MVC) method. Embryos were equilibrated with equilibration solution containing 7.5% (v/v) ethylene glycol (EG), 7.5% (v/v) dimethylsulfoxide (DMSO), and 20% (v/v) calf serum for 4 min, followed by exposure to vitrification solution containing 15% EG, 15% DMSO, 0.5 M sucrose, and 20% calf serum. Embryos were then loaded onto a Cryotop (Kitazato Supply Co., Tokyo, Japan) and immediately plunged into liquid nitrogen. Vitrified embryos were examined for viability in vitro and in vivo after warming. Their in vitro developmental competence was compared to that of corresponding control (nonvitrified) embryos. Vitrified 4- to 8-cell stage embryos, both parthenogenetic and IVF, showed developmental competence into blastocysts comparable to that of control embryos (parthenogenetic: 46.8%, 36/77 vs. 51.7%, 31/60; IVF: 40.0%, 30/75 vs. 44.3%, 35/79). Of four surrogate gilts that received a total of 251 vitrified parthenogenetic embryos, three became pregnant and had 20 fetuses (8.0%, 22 to 23 days old). Three surrogates gilts that received 267 vitrified IVF embryos all became pregnant. Of those, the one that received 47 embryos was confirmed to have eight fetuses (17.0%, 22 days old) by autopsy. The other two were examined by ultrasonography at 56 and 95 days of gestation and found to be pregnant. These results suggest that porcine embryos derived from IVM oocytes have a potential to develop into live offspring after delipation and MVC vitrification. This study was supported by PROBRAIN.

88 PREGNANCY RATES FOR IN VITRO AND IN VIVO PRODUCED OVINE EMBRYOS VITRIFIED USING THE MINIMUM VOLUME COOLING CRYOTOP METHOD

Previously we reported that, using the minimum volume cooling (MVC) cryotop vitrification method, in vitro-produced ovine and bovine embryo survival after thawing was similiar to that of fresh embryos (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172). While survival of vitrified embryos after thawing can be indicative of embryo viability, this assessment does not always correlate with embryo survival after transfer. This study assesses the effect of vitrification using the MVC cryotop method on the survival after transfer of in vitro- and in vivo-produced ovine embryos. Fresh or vitrified Day 6 ovine embryos (expanded blastocysts, blastocysts, compact morulae) were used in this study. Ovine cumulus–oocyte complexes were obtained and matured, fertilized (Day 0), and cultured in vitro (Walker et al. 1996 Biol. Reprod. 55, 703–708). In vitro embryos for vitrification were produced and vitrified (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172) 10 days prior to the day of transfer. In vivo embryos were recovered from donor Merino ewes and vitrified 7 days prior to the day of transfer while fresh in vivo embryos were collected and transferred on the same day. Semen used for both in vivo and in vitro embryo production was from the same sire. On the day of transfer, vitrified embryos were thawed directly into 1.25 M sucrose solution, followed by stepwise dilution of the cryoprotectants. Embryos were transferred as singles into synchronized recipient ewes on a randomized basis. Fetal number was detected at Day 50. Variables were assessed using the CATMOD procedure in SAS. Pregnancy rate for in vivo-derived embryos was higher (P &lt; 0.01) than for in vitro-derived embryos. Embryo treatment (fresh vs. vitrified) did not significantly affect pregnancy rate. Pregnancy rate for ewes detected (by vasectomized rams) in estrus within 48 h of progesterone pessary removal was higher (P &lt; 0.05) than for both the 48–68 h and unmarked groups. The latter two groups did not differ significantly. None of the first-order interactions were significant (P &gt; 0.05). This study demonstrates that ovine embryos (in vitro and in vivo) can be vitrified, thawed, and transferred without compromising embryo viability. However, the differences in pregnancy rate between the recipient groups warrant further investigation. The MVC cryotop method is a vitrification technique that can be adapted to routine field use. Table 1. Pregnancy rate of fresh and vitrified in vivo and in vitro ovine embryos after embryo transfer

Pregnancies resulting from transfer of human blastocysts vitrified by the minimum-volume-cooling method

The purpose of this study was to evaluate the clinical efficacy of the minimum-volume-cooling (MVC) method to cryopreserve human blastocysts by vitrification. We have used this method before to vitrify oocytes, producing term pregnancies by transfer of embryos derived by ICSI of vitrified oocytes (2003 Fertil Steril 80:223).

Cryopreservation of porcine embryos derived from in vitro-matured oocytes.

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.

102VITRIFICATION OF IN VITRO-PRODUCED BOVINE AND OVINE EMBRYOS USING THE MINIMUM VOLUME COOLING CRYOTOP METHOD

Considerable progress has been achieved in the cryopreservation of mammalian embryos. The use of vitrification minimizes chilling injuries by increasing cooling and warming rates. This study assesses the effect of vitrification using the minimum volume cooling (MVC) method (Kuwayama &amp;amp; Kato 2000 J. Assist. Reprod. Genet. 17, 477) on in vitro-produced bovine and ovine embryos. A total of 1756 ovine and 753 bovine cumulus-oocyte complexes were obtained from the abattoir and matured, fertilized (Day 0) and cultured in vitro (Walker et al., 1996 Biol. Reprod. 55, 703–708, Kelly et al., 1997 Theriogenology 47, 291). Overall cleavage rates were 93.7% and 80.5% respectively. Embryos were vitrified (OPS or MVC method) on Days 5 (morula, compact morula), 6 (expanded blastocyst, blastocyst, compact morula) or 7 (hatched and hatching blastocysts, expanded blastocyst, blastocyst). Embryos were equilibrated with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 3min and then exposed to 16.5% EG, 16.5% DMSO, 0.5M sucrose and 20% FCS for 30s. Embryos were loaded onto either an MVC plate (Cryotop, Kitazato Supply Co, Toyko, Japan) or open pulled straw (OPS) and plunged into liquid nitrogen. After 5 days, embryos were thawed directly into 1.25M sucrose solution at 38.5°C, followed by stepwise dilution of the cryoprotectants. Embryo survival was assessed by culture to Day 8 and compared to the development of non-vitrified control embryos (Table 1). Variables were assessed using procedure CATMOD in SAS. The Cryotop method yielded a significantly higher percentage of viable ovine embryos after thawing compared with OPS (P&amp;lt;0.0001); neither day nor treatment x day interaction was significant (P&amp;gt;0.05). A significant interaction between vitrification treatment and day (P&amp;lt;0.007) indicated that the percentage of hatched embryos peaked at Day 6 using the Cryotop method compared with Day 7 for OPS. Hatching rates for fresh and vitrified embryos were similar at Day 7 and were independent of treatment. With the Cryotop method, day of vitrification did not influence the percentage of Days 6 and 7 bovine embryos that hatched after thawing but, on each day, this figure was significantly higher (P&amp;lt;0.003 and P&amp;lt;0.0001, respectively) than that obtained with fresh embryos. To further assess embryo viability, 36 fresh, 52 OPS and 56 Cryotop vitrified Day-6 in vitro-produced ovine embryos were transferred to synchronized recipients. Survival rates to Day 13 were 29/33 (87.9%), 23/36 (63.9%) and 42/51 (82.4%), respectively (P&amp;lt;0.05). This study demonstrates that using the MVC Cryotop method, the viability of vitrified embryos, as assessed at Days 8 and 13, is similar to that obtained with fresh embryos. Table 1

Improved survival of vitrified in vivo-derived porcine embryos.

An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 microg/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +/- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +/- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.