Comparison of DNA apoptosis in mouse and human blastocysts after vitrification and slow freezing

Vitrification is a novel cryopreservation method for mammalian blastocysts. This study was designed to compare different vitrification methods and slow freezing for their effects on survival rate and DNA integrity in mouse and human blastocysts. In Experiment 1, embryo survival and DNA integrity were compared between mouse blastocysts with collapsed and non‐collapsed blastoceles. In Experiment 2, embryo survival and DNA integrity were compared between vitrified and slow‐frozen mouse blastocysts. In Experiment 3, embryo survival and DNA integrity were compared between vitrified and slow‐frozen human blastocysts. Fresh blastocysts were used as controls in all experiments. Higher (P < 0.05) blastocyst survival rates were obtained in mouse blastocysts vitrified with collapsed versus intact blastoceles, although DNA‐integrity indices in the surviving blastocysts were the same among vitrified and fresh blastocysts. More mouse blastocysts (P < 0.05) survived after vitrification (100%) as compared to slow freezing (82.5%). DNA‐integrity indices examined in the surviving blastocysts were also higher (P < 0.001) in fresh (93.6%) and vitrified/warmed (93.7%) blastocysts than in slow‐frozen/thawed (75.8%) ones. More human blastocysts survived with a higher DNA‐integrity index after vitrification/warming than after slow freezing/thawing. These results indicate that higher survival rates can be obtained by vitrification of blastocele‐collapsed blastocysts, and that vitrification causes less cell apoptosis in both mouse and human blastocysts compared to slow freezing. Vitrification of blastocysts after blastocele collapse by single laser pulse supports a higher survival rate and less DNA apoptosis, suggesting that laser blastocele collapse is a safe procedure for blastocyst vitrification. Mol. Reprod. Dev. 79: 229–236, 2012. © 2011 Wiley Periodicals, Inc.     

Cryopreservation of mouse embryos by vitrification: A meta-analysis

A meta-analysis of cryopreservation studies vitrifying mouse embryos was undertaken to determine the treatment effect of vitrification. Treatment by vitrification decreased embryo viability compared with controls: the odds ratio was 9.02 (CI: 3.73-21.78; P < 0.001), a 24.90% (CI: 14.88-34.91; P < 0.001) reduction in risk was associated with embryos in the control group, and for every 4.00 (CI: 3.91-4.09) embryos treated by vitrification, one does not survive. A multiple regression analysis evaluated covariates of embryo survival. For each hour increase post-hCG treatment when embryos were cryopreserved, there was a decrease of 0.36% (SEM ± 0.01) in survival (P < 0.001). The number of embryos surviving vitrification decreased 0.25% (SEM ± 0.02) per day increase in age of the female mouse (P < 0.001), whereas there was no significant difference for control group embryos. For each 1 h increase post-hCG treatment after cryopreservation when blastocysts were assessed for viability, there was a decrease of 0.13% (SEM ± 0.01) in survival. The later interval post-hCG treatment when blastocysts were assessed, the less viable they were compared with earlier blastocysts, independent of the vitrification protocol. This effect was not observed for control embryos. A high percentage of variability in the treatment effect for vitrification was likely due to underlying heterogeneity among studies. A portion of the risk associated with vitrification could be attributed to the general effects of cryopreservation. Future research should identify effects in a cryopreservation protocol specific to vitrification that affect viability of mouse embryos.     

Some factors affecting successful vitrification of mouse blastocysts

The effects of temperature and exposure time to vitrification solutions on In vitro survival of mouse blastocysts were investigated. Blastocysts were first exposed for 10 min to vitrification Solution 1 (VS1) containing 10% glycerol-20% 1,2 propanediol in phosphate buffered saline (PBS), then to vitrification Solution 2 (VS2) with 25 % glycerol-25% 1,2 propanediol for various periods either at room temperature or at 4°C. At room temperature survival dropped quickly, while at 4°C an increase in survival was observed.

It is concluded that the viability of mouse blastocyts after vitrification is dependent on the temperature and duration of equilibration in vitrification solutions.     

Comparison of open pulled straw (OPS) vs glass micropipette (GMP) vitrification in mouse blastocysts

The purpose of this study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of mouse blastocysts, and to compare the post-thaw survival of these blastocysts with those cooled in open pulled straws (OPS). The GMP vessel permits higher freezing and warming rates than OPS due to the higher heat conductivity of glass and lower mass of the solution containing the embryos. Groups of 6 mouse blastocysts were sequentially placed into 2 vitrification solutions before being loaded into either the OPS or GMP vessels and immersed into LN2 within 20 to 25 sec. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in holding medium (HM) and modified human tubal fluid medium (mHTF), each for 5 min, and then cultured in mHTF supplemented with 10% FCS for 24 h. The rate of blastocyst re-expansion did not differ significantly for OPS (93.5%) and GMP (95.0%) methods (P<0.05). The hatching rate in OPS (88.7%) was similar to that in GMP (90.0%) but was lower than for the unvitrified control embryos (98.3%, P<0.05). To determine the optimal embryo population per GMP vessel, the pipettes were loaded with 2 to 10 embryos. The rate of blastocyst re-expansion after vitrification was significant for 2 to 4 embryos than for 6 to 10 embryos per vessel. In addition, the rate of blastocyst re-expansion was significantly lower if blastocysts were vitrified in the wide rather than the narrow portion of the micropipette (100 vs 87.5%; P<0.05) even when only 4 blastocysts were loaded per vessel. These results indicate that both vitrification vessels can provide high rates of embryo survival. However, the GMP vessel does not need a cap to protect the vessel from floating after immersion in LN2. The number and location of the embryos (narrow versus wide portion of capillary) were considered to be limiting factors to the viability of mouse embryos.     

High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival

An innovative technique, called the high hydrostatic pressure (HHP) treatment, has been recently reported to improve the cryosurvival of gametes or embryos in certain mammalian species. The aim of the present study was to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups, respectively) before they were used for HMC. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method based on minimum volume cooling procedure. The blastocyst rate was higher in the HHP2 group than in the control group (68.2 +/- 4.1% vs. 46.4 +/- 4.2%; p < 0.01), while there was no difference between HHP1 and control group (52.1 +/- 1.2% vs. 49.0 +/- 2.7%; p > 0.05). Similar mean cell numbers of produced blastocysts were obtained in HHP2 and control groups (56 +/- 4 vs. 49 +/- 5; p > 0.05). Subsequent blastocyst vitrification with the Cryotop method resulted in significantly higher survival rate after thawing in the HHP2 group than in the control group (61.6 +/- 4.0% vs. 30.2 +/- 30.9%; p < 0.01). Fifty-six and 57 day 5 to day 7 fresh blastocysts in HHP1 group were transferred into two recipient sows on day 5 of the estrous cycle. One recipient was diagnosed pregnant and gave birth to two healthy piglets by naturally delivery on day 122 of gestation. This pilot study proved that the sublethal HHP treatment of porcine oocytes before HMC results in improved in vitro developmental competence and cryotolerance, and supports embryonic and fetal development as well as pregnancy establishment and maintenance up to the birth of healthy piglets.     

The effect of equilibration time on survival and development rates of mouse pronuclear-stage embryos vitrified in solid surface (SSV) and convential straws: in vitro and in vivo evaluations

The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage (PN) mouse embryos. A novel vitrification technique (solid surface vitrification, SSV) was compared with a convential one in straws both for cryosurvival and obtaining progeny from cryopreserved PN mouse embryos. In the SSV method, 15-20 PN embryos were exposed to vitrification solutions for approximately 20 sec after equilibration, and then they were dropped in 2 microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. In the straws method, groups of 5-10 PN embryos were loaded in a single straw after equilibration. In experiment I, it was compared the effect of the vitrification solutions alone, without vitrification. No reduction was detected in survival, cleavage and blastocysts rates and the lowest development rate was obtained from hatched blastocyst for 20 min equilibration (24.5%). In experiment II, SSV method resulted in significantly higher survival and cleavage rates than that of in-straw vitrified 15-20 min group (87% vs. 60%, 83% vs. 67%, respectively; P < 0.05). There were no statistical differences among any of the blastocyts groups. However, there was a statistical difference in hatched blastocysts between 15 to 5, 10, and 20 min (P < 0.05). In experiment III, it was found no major effect among equilibration time periods in toxicity groups according to the mean cell number of blastocysts developed from PN embryos. But, there was a significant differences between 15 min SSV and 10 min in straw vitrified according to the mean cell number of blastocysts developed from PN embryos following vitrification (P < 0.05). The good results were obtained from 15 min equilibration group for SSV and 10 min equilibration group for straw vitrification. In the last experiment, embryo transfer after vitrification and toxicity was investigated. There were significant differences between SSV and straw just on the rate of pups born (30% and 20.5% respectively; P < 0.05). In conclusion, vitrification of PN mouse embryos by SSV can result in high rates of in vitro development to expanded and hatched blastocyst stage and in vivo development to live pups.     

Carboxylated ε-poly-L-lysine,a cryoprotective agent,is an effective partner of ethylene glycol for the vitrification of embryos at various preimplantation stages

It has been known that different protocols are used for embryo preservation at different stages due to different sensitivity to the physical and physiological stress caused by vitrification. In this study, we developed a common vitrification protocol using carboxlated ε-poly-l-lysine (COOH-PLL), a new cryoprotective agent for the vitrification of mouse embryos at different stages. The IVF-derived Crl:CD1(ICR) x B6D2F1/Crl pronuclear, 2-cell, 4-cell, and 8-cell, morula and blastocyst stage embryos were vitrified with 15% (v/v) ethylene glycol (EG) and 10% (w/v) COOH-PLL (E15P15) or 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (E15D15) using the minimal volume cooling method. The survival of vitrified embryos from pronuclear to blastocyst stages was equivalent between E15P15 and E15D15 groups. However, the rate of development to blastocysts was significantly lower in E15P15 than E15D15. The rates of survival and development to blastocysts were dramatically improved by a slight modification of EG and COOH-PLL concentrations (E20P10). After transferring 17 (E20P10) and 15 (E15D15) vitrified/warmed blastocysts, 8 and 7 pups were obtained (47.1% and 46.7%, respectively). Taken together, these results indicate that our vitrification protocol is appropriate for the vitrification of mouse embryos at different stages.     

Metal mesh vitrification (MMV) method for cryopreservation of porcine embryos

The objective was to develop a simpler, more reliable vitrification method for porcine embryos. Prepubertal donor gilts were induced to ovulate with eCG and hCG, and then inseminated artificially. Morulae and expanding blastocysts approximately 200 microm in diameter were collected 6 or 7d after hCG treatment. Embryos collected from donor gilts were maintained, so as to be individually recognizable, and handled in batches of four or five. The embryos together with a minimum volume (<2 microL) of vitrification solution were placed onto stainless steel metal meshes or plastic plates, and then plunged into liquid nitrogen-metal mesh vitrification (MMV) and plastic plate vitrification (PPV), respectively. The meshes or plates were stored in 1.8-mL cryotubes submerged in liquid nitrogen. Stored embryos were subsequently removed, cultured in medium for 24 h, and then assessed for viability. The survival rate (84.4%) of expanding blastocysts cooled by MMV was higher than that (53.1%) of embryos cooled by PPV (P<0.05). There was no significant difference in total cell number between MMV and PPV. The survival rate of morulae cooled by MMV was 55.0%. Transfer of 200 expanding blastocysts cooled by MMV to 10 synchronized recipient gilts resulted in 37 live piglets from 7 recipients. In conclusion, the MMV method was an effective vitrification procedure for cryopreservation of expanding porcine blastocysts. However, there was a batch effect on embryo survival after vitrification.     

Bovine blastocyst development in vitro: timing, sex, and viability following vitrification

Selection of blastocysts based on their morphological characteristics and rate of development in vitro can skew the sex ratios. The aim of this study was to determine whether an embryo's developmental rate affects its survival after vitrification, and whether male and female embryos survive vitrification differently. In vitro fertilized bovine oocytes were cultured in potassium simplex optimized medium (KSOM) + 0.1% BSA for 96 h, and then into KSOM + 1% BSA (KSOM) or in sequential KSOM + 0.1% BSA for 96 h, and then into synthetic oviduct fluid (SOF) + 5% FBS (KSOM-SOF). In part 1 of this study, embryos cultured in each medium that had developed into blastocysts at approximately 144, 156, or 180 h were recovered from culture, graded, and then vitrified. After warming, blastocyst survival rates were immediately evaluated by reexpansion of the blastocoels. In the second part of the study, all blastocysts (n = 191) were sexed by polymerase chain reaction 48 h after warming. When cultured in KSOM medium, more 144-h blastocysts survived vitrification (68%) than blastocysts vitrified at 180 h (49%). Blastocysts derived at 156 h in KSOM-SOF survived vitrification better (87%) than blastocysts vitrified at either 144 h or 180 h, and subsequently hatched at a greater rate than those vitrified at 180 h. The overall blastocyst survival rates did not differ significantly whether embryos were cultured in KSOM or sequential KSOM-SOF. Blastocysts derived at 144 and 156 h in KSOM or KSOM-SOF were predominately male, and significantly more of them survived vitrification 48 h after warming. However, blastocysts cultured in KSOM-SOF, and then vitrified at 180 h were predominately female. Overall, blastocysts that survived vitrification, and subsequently hatched 48 h after warming, were male. In summary, embryos that reached the blastocyst stage earlier were predominantly males; these males had better morphology, endured vitrification, and subsequently hatched at a greater rate than did female blastocysts.     

Effects of equilibration time, precooling and developmental stage on the survival of mouse embryos cryopreserved by vitrification

Factorial experiments were carried out to examine the effects of equilibration time, precooling and developmental stage on the postthaw in vitro survival of vitrified mouse embryos. Eight-cell embryos, compacted morulae, or blastocysts were cryopreserved using vitrification Solution 1 (VS1; 10% glycerol + 20% propylene glycol), and vitrification Solution 2 (VS2; 25% glycerol + 25% propylene glycol) in phosphate buffered saline + 10% calf serum. Each embryo stage group was first equilibrated in VS1 for 5, 10 or 20 min and then exposed to either a precooled ( approximately 4 degrees C) or nonprecooled ( approximately 20 degrees C) VS2 in a 0.25-ml straw before they were plunged directly into liquid nitrogen. Results of this study showed an interaction between precooling, equilibration time and developmental stage which affect significantly post-thaw embryo survival (P< 0.05). High survival rates were obtained after 10 min equilibration in VS1 irrespective of the embryo developmental stage. Precooling of the VS2 significantly improved the survival mainly of blastocysts. However, eight-cell and morula-stage embryos also showed high survival rates when they were exposed to precooled VS2 after 5 min equilibration in VS1. It was further observed that morulae usually exhibit high survival rates, and vitrification conditions are more critical for early and advanced stage embryo development.     

Aberrant expression of miR-29a/29b and methylation level of mouse embryos after in vitro fertilization and vitrification at two-cell stage

Proper epigenetic modifications during preimplantation embryo development are important for a successful pregnancy. We aim to investigate the putative influence of in vitro fertilization (IVF) and vitrification on DNA methylation in mouse preimplantation embryos. The study groups consisted of blastocyst-derived vitrified two-cell embryos, nonvitrified embryos, and a control group of in vivo derived blastocysts. We assessed developmental competence, global DNA methylation, relative expression levels of miR-29a/29b, and their target genes, Dnmt3a/3b. Vitrified embryos had a lower developmental rate as compared with nonvitrified embryos. There was no significant decrease in blastocyst cell numbers among studied groups, whereas there was a steady decline in DNA methylation after IVF and vitrification. The levels of miR-29a/29b upregulated in the experimental groups as compared with the control group. IVF and vitrification caused Dnmt3a/3b downregulations in blastocysts. The results of this study have suggested that a relationship exists between IVF and embryo vitrification with methylation interruptions in the blastocysts.     

Vitrification of mouse embryos in two cryoprotectant solutions

The objective of this study was to compare the efficiency of 2 media on the vitrification of mouse compacted morulae, early blastocysts and expanded blastocysts after equilibration at room temperature of 4 degrees C. Embryos were equilibrated for 10 min in either 25% VS3 (Rall Equilibration Medium, REM) or 10% glycerol + 20% propylene glycol (Massip Equilibration Medium, MEM) in DPBS at 20 degrees C or 4 degrees C. For vitrification either 100% VS3 (Rall Vitrification Medium, RVM) or 25% glycerol + 25% propylene glycol (Massip Vitrification Medium, MVM) in DPBS was used. Embryos equilibrated at room temperature were loaded in 20 microL of vitrification media into 250 microL straws and then immediately (30 sec) plunged into liquid nitrogen (LN2). After equilibration at 4 degrees C the embryos were put into straws with 20 microL of precooled vitrification medium, and after 20 min at 4 degrees C they were plunged into LN2. Embryos from both groups were thawed in a 20 degrees C water bath for 20 sec, transferred to 1.0 M sucrose in DPBS for 5 min and then cultured for 24 to 48 h in Whitten's medium at 37 degrees C in 5% CO2 in air. In the groups of embryos prepared for vitrification at room temperature the survival rate of compact morulae vitrified in RVM was higher than those vitrified in MVM (65/70, 93% vs 49/74, 66%; P < 0.01). No difference was found in the survival rate of early blastocysts and expanded blastocysts vitrified in RVM or MVM (30/83, 36% vs 25/75, 33% and 4/66, 6% vs 4/76, 5%). No difference was found between the survival rate of compact morulae after equilibration with RVM or MVM at 4 degrees C (62/75, 83% vs 52/74, 70%). Both the early blastocysts and expanded blastocysts equilibrated at 4 degrees C MVM yielded a higher survival rate than RVM (28/74, 38% and 40/70, 57% vs 4/75, 5% and 4/77, 5%; P < 0.01). We conclude that, of the 3 developmental stages, compact morulae withstand the vitrification process best, and reduction of the temperature prior to plunging into LN2 is not required. A 10-fold increase in the survival rate of expanded blastocysts can be achieved using low temperature equilibration (4 degrees C) and MVM.     

Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of vitrified in vitro sheep embryos

Embryo cryopreservation is an important tool to preserve endangered species. As a cryoprotectant for mouse oocytes, antifreeze protein from Anatolica polita (ApAFP914) has demonstrated utility. In the present study, the effects of controlled slow freezing and vitrification methods on the survival rate of sheep oocytes fertilized in vitro after freezing-thawing were compared. Different ApAFP914 concentrations were added to the vitrification liquid for exploring the effect of antifreeze protein on the warmed embryos. The results showed that the survival and hatching rates of in vitro derived embryos were significantly higher than that of the slow freezing method. Furthermore, among the cryopreserved embryos at different developmental stages, the survival and hatching rates of the expanded blastocyst were significantly higher than those of the blastocysts, early blastocysts and morula. The survival and the hatching rates of the fast-growing embryos were both significantly higher than that of the slow-growing embryos. Additionally, treatment of ApAFP914 (5–30 μg/mL) did not increase the freezing efficiency of the 6–6.5 d embryos. However, addition of 10 μg/mL of ApAFP914 significantly increased the hatching rate of slow-growing embryos. In conclusion, our study suggests that the vitrification is better than the slow freezing method for the conservation of in vitro sheep embryos, and supplementation of ApAFP914 (10 μg/mL) significantly increased the hatching rate of slow-growing embryos after cryopreservation.     

Open pulled straw vitrification of goat embryos at various stages of development

This investigation addresses the question whether it is possible to apply the open pulled straw (OPS) vitrification method, found to be effective for cryopreserving caprine (Capra aegagrus hircus) blastocysts, to other embryonal stages. Morulae, blastocysts and hatched blastocysts were cryopreserved by way of OPS vitrification and blastocysts and hatched blastocysts by conventional freezing. Morulae were not included with conventional freezing because in our experience the survival rate is very low. To assess the viability of the cryopreserved embryos, they were transferred to synchronized does; in most cases, two embryos per doe. After OPS vitrification, of nine does receiving morulae, not a single one became pregnant; of 11 does receiving blastocysts, nine (82%) became pregnant (all of which kidded and gave birth to, on average, 1.8 kids); and of nine does receiving hatched blastocysts, three (33%) became pregnant (two of which [22%] kidded, giving birth to a single kid each). After conventional freezing, of 10 does receiving blastocysts, five became pregnant (four of which [40%] carried to term and gave birth to a pair of twins each); and of nine does receiving hatched blastocysts, three (33%) became pregnant (and gave birth to a single kid each). Embryo survival (kids born/embryos transferred) after vitrification for morulae, blastocysts, and hatched blastocysts was 0, 70% (16 of 23), and 13% (2 of 16), respectively, and after conventional freezing for blastocysts and hatched blastocysts was 42% (8 of 19) and 19% (3 of 16), respectively. The difference in pregnancy and kidding rate between vitrified and conventionally frozen blastocysts was significant, and so was the difference in pregnancy rate between hatched and nonhatched blastocysts, regardless whether OPS-vitrified or conventionally frozen. The results of the current study indicate that OPS vitrification is a very effective means of cryopreserving caprine blastocysts. Unfortunately, the superiority of OPS vitrification over conventional freezing does not apply to caprine morulae and hatched blastocysts.     

Vitrification of Mouse Embryos at Various Stages by Open-Pulled Straw (OPS) Method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure—that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method—that is, embryos were first pretreated in 10%E+10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E+10%D for 0.5 min, exposed to EDFS30 for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: effect of developmental stage and age

The objective was to investigate the effects of developmental stage (fully-expanded or expanding blastocysts) and/or age (harvested on Days 7 or 8) on post-vitrification in vitro survival of bovine blastocysts derived from intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Post-warming survival (re-expansion of blastocoele within 24 h) of ICSI-derived fully-expanded blastocysts (80%) was similar to that of their IVF-derived counterparts (88%). However, the ability of ICSI-derived expanding blastocysts to survive vitrification procedures (61%) was lower than that of IVF-derived blastocysts (85%; P < 0.05), although the ICSI- and IVF-derived fresh blastocysts were of similar quality. The age of the blastocysts before vitrification did not affect cryotolerance for either ICSI-derived (73 and 59% for Days 7 and 8 embryos, respectively) or IVF-derived blastocysts (86% for both Days 7 and 8 embryos). At 24 h of post-warming culture, ICSI-derived blastocysts surviving vitrification contained a higher proportion of dead cells than their IVF-derived counterparts (5-13% vs. 2-4%; P < 0.05), but these proportions were not different from those of fresh control embryos. There was an adverse effect of vitrification on the ability of blastocysts to hatch within 72 h of culture only in IVF-derived Day 8 blastocysts (41 and 70% in vitrified and fresh control groups, respectively). In conclusion, the proportion of blastocysts that survived vitrification procedures was similar for ICSI- and IVF-derived bovine blastocysts if the former were cultured to the fully-expanded stage prior to vitrification, with no significant difference between embryos harvested on Day 7 versus Day 8.     

Vitrification of mouse embryos at various stages by open-pulled straw (OPS) method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure-that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method-that is, embryos were first pretreated in 10%E + 10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E + 10%D for 0.5 min, exposed to EDFS30for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

不同品系小鼠胚胎玻璃化冷冻保存的比较研究

目的　研究甘油作为冷冻保护剂、不同基因型小鼠对胚胎玻璃化冷冻的影响。方法　采用 6 5mol L的甘油作为冷冻保护剂 ,采用二步法对CBA、NOD、C57BL 6J、ICR及CD1小鼠 3 5d的胚胎进行玻璃化冷冻 ,并比较了不同品系小鼠胚胎的复苏率及移植受孕率。结果和结论　CBA、NOD、C57BL 6J,ICR及CD1的复苏率分别为 5 7 6 %、4 8%、31 3%、86 5 %及 88% ,移植受孕率为 2 1%、2 3 5 %、11%、38%和 35 5 % ,封闭群小鼠的胚胎复苏率、移植受孕率均显著高于近交系小鼠。这提示胚胎的复苏率及移植受孕率可能与小鼠的不同基因型有关。五个品系中 ,桑椹胚及早期囊胚的体外复苏率均显著高于扩张囊胚。这说明不同基因型及胚胎的不同发育阶段对胚胎玻璃化冷冻效果有影响     

A new vitrification device that absorbs excess vitrification solution adaptable to a closed system for the cryopreservation of mouse embryos

Several closed vitrification devices that avoid contact with liquid nitrogen have been reported. Recently, based on the Kitasato Vitrification System (KVS), we developed the Closed-KVS, which is a closed vitrification device. The KVS is an open vitrification device that can absorb excess vitrification solution. In this study, we performed two experiments to evaluate the efficacy of the Closed-KVS as a vitrification device for the cryopreservation of mouse embryos at the blastocyst and two-cell stage. In the first experiment, the blastocysts were vitrified using either the Closed-KVS or the KVS (control device). The survival, re-expansion, and hatching rates were not significantly different between embryos vitrified using the Closed-KVS and those vitrified using the KVS. In the second experiment, we evaluated the embryonic development of the two-cell stage embryos vitrified using the Closed-KVS. There were no significant differences in the survival, blastocyst formation, or hatching rates between vitrified or non-vitrified embryos. Additionally, we evaluated the cooling and warming rates of these devices using a numerical simulation method. The cooling rates of the Closed-KVS were similar regardless of whether the outer cap was pre-cooled and were lower than those of the KVS. However, the warming rates of the Closed-KVS (irrespective of cap pre-cooling) were the same as those of the KVS (612,000 °C/min). In summary, the Closed-KVS is a novel closed vitrification device for the cryopreservation of mouse embryos at the blastocyst and two-cell stage.