Post-thaw survival, cell death and actin cytoskeleton in gene-microinjected rabbit embryos after vitrification

The aim of this study was to compare two vitrification procedures (VPs), using either ethylene glycol (EG) in combination with dimethylsulfoxide (DMSO, vitrification protocol (VPI)) or Ficoll 70 (vitrification protocol II (VPII)), for rabbit embryo cryopreservation based on their post-thaw survival, cell death and actin cytoskeleton. The pronuclear stage eggs were flushed from the oviducts of the slaughtered New Zealand White rabbit does 19-20h post coitum (hpc) and randomly divided into two groups: intact (control) and microinjected (Mi). Mi embryos or intact embryos were cultured for up to 72hpc (morula stage), and then vitrified using either VPI (VPI+Mi, VPI) or VPII (VPII+Mi, VPII). After 2-3 days at -196 degrees C, the embryos were thawed and cultured until 96-100hpc to assess their development to blastocyst, apoptotic rate (TUNEL assay) and state of actin cytoskeleton (phalloidine-TRITC). Mi procedure reduced blastocyst yield, but it was higher than in either vitrified (VPI) or Mi vitrified (VPI+Mi) embryos. VPI compromised, whereas VPII did not significantly affect blastocyst development compared to intact embryos. Mi and VP both affected the embryo quality increasing TUNEL-index and decreasing the ratio of embryos with high quality actin cytoskeleton compared to control. A higher apoptotic index was recorded in VPI group. A combination of Mi and VP induced an increase in apoptotic rate (10.35% and 7.54% for VPI+Mi and VPII+Mi, respectively) as compared to Mi alone (5.7%). Ratio of embryos belonging to best actin quality (grade I) was different among groups and most of the embryos with grade I actin were in intact (84%), Mi (71%) or VPII (70%) groups. A significantly lower number of embryos with grade I actin quality was observed in VPI (58%), VPI+Mi (54%) or VPII+Mi (66%). These observations indicate that of the vitrification schemes tested, the VPII using EG and ficoll 70 as cryoprotectants, was less harmful than VPI (EG combined with DMSO in vitrification medium).     

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

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

In vitro postwarming viability of vitrified porcine embryos: Effect of cryostorage length

Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN₂) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN₂ has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage.     

Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method

Mouse oocytes and embryos at various developmental stages were exposed directly to an ethylene glycol-based vitrification solution (EFS) for 2 or 5 minutes at 20 degrees C. They were then vitrified at -196 degrees C and were warmed rapidly. At the germinal vesicle stage, the proportion of morphologically normal oocytes was 36 to 39% if they had cumulus cells, whereas in cumulus-removed immature oocytes and in ovulated oocytes it was only 2 to 4%. This low survival was attributed to the harmful action of ethylene glycol. After fertilization, on the other hand, the post-warming survival rate of 1-cell zygotes, as assessed by cleavage to the 2-cell stage, increased markedly (62%). As the developmental stage proceeded, higher proportions of vitrified embryos developed to expanded blastocysts; the rates increased up to 77 and 80% in 2-cell and 4-cell embryos, respectively. For embryos at the 8-cell, morula and early blastocyst stages, the proportion of embryos developed after vitrification (90 to 95%) was not significantly different from that of the untreated embryos (95 to 100%) when the period of exposure to EFS solution was 2 minutes. As the blastocoel began to enlarge, however, survival began to decrease again, with rates of 79 and 57% in blastocysts and expanded blastocysts, respectively. After the cryopreserved 2-cell, 4-cell and 8-cell embryos as well as morulae and blastocysts were transferred to recipients, 43 to 57% of the recipients became pregnant, and 48 to 60% of these various stage embryos developed into live young.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

New device for the vitrification and in-straw warming of in vitro produced bovine embryos

Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1, 0.5 and 0 M sucrose; protocol W3) or directly in 0.5 M (protocol W1/0.5) or 0 M sucrose (protocol W1/0). Similar survival rates were recorded 24 h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24 h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5 M sucrose) dilution/warming and different warming temperatures (45, 50, 60 and 70 °C) were tested. When warming solution passed through the new vitrification/warming device at 45 °C, 61.5% of blastocysts were fully re-expanded or hatched at 24 h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24 h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45 °C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions.     

Banking of embryos of mutated, paralytic tremor rabbit by means of vitrification

Cryopreservation enables banking of embryos for future use in medicine and in animal breeding. It also enables protection of germ plasm of endangered species and unique strains or lanes of laboratory animals. This paper describes an example of employing a vitrification method for banking of embryos of a unique lane of rabbit. The paralytic tremor (pt) rabbit is an X-linked recessive mutant lane of the Chinchilla breed characterized by hypomyelination of the central nervous system. In order to obtain a sufficient number of embryos, pt females were subjected to superovulation and surgical embryo collection. All suitable embryos were vitrified in 0.25 mL insemination straws in a modified EFS vitrification solution comprised of ethylene glycol (40%), Ficoll 70 (18%) and sucrose (0.3 M) in Hepes buffered TCM medium containing 20% fetal calf serum. In order to assess the efficiency of the vitrification procedure, a representative portion of vitrified embryos was warmed after a period of storage. Warmed embryos were subjected to in vitro culture for 72 h or were transferred to the uterus of synchronized recipients. The majority of the 141 warmed embryos survived vitrification and 100/141 (71%) developed to the blastocyst stage. Moreover, out of an additional 34 warmed embryos transferred to four recipients, eight (23.5%) developed to term and seven live pups were born. Six of the rabbit pups exhibited paralytic tremor symptoms typical for the pt lane. Although the overall efficiency of the vitrification method was lower compared with the effects usually achieved for 'healthy' embryos, results presented confirm the real possibility of the future restoration of the colony of pt rabbit, if sufficient number of embryos are cryopreserved.     

Ultra-structural changes and developmental potential of porcine oocytes following vitrification

This study evaluated the effects of exposure and/or vitrification of porcine metaphase II (MII) oocytes on their in vitro viability and ultra-structural changes with two experiments. Experiment 1 examined the effect of vitrified oocytes on microtubule localization, mitochondrial morphology, chromosome organization and the developmental rate in IVF control and vitrified oocytes. Oocytes matured for 44 h were subjected to IVF (IVF control). Oocytes matured for 42 h were exposed to cryoprotectants (CPA control), followed by 2h culture, and subjected to IVF. Oocytes vitrified at 42 h post-maturation were warmed, cultured for 2h, and subjected to IVF (vitrified). Experiment 2 evaluated the effect of oocytes freezing on development of ICSI with and without activation and parthenotes. Fresh and vitrified oocytes were subjected to ICSI with and without electrical activation. Cleavage and blastocyst rates were significantly (P<0.05) lower in vitrified IVF, parthenote and ICSI embryos than those in fresh counterparts. Between ICSI embryos from fresh oocytes and vitrified oocytes, the rates of blastocyst were significantly higher (P<0.05) in activated group than the group without activation. Significant differences (P<0.05) were observed in normal spindle configuration of vitrified (43.5%) compared to control (81.0%) oocytes, but no significant difference was observed between CPA exposed and control groups. In conclusion, porcine oocytes at MII stage are very sensitive to vitrification with altered microtubule localization and mitochondrial organization thus resulting in impaired fertilization and embryo development.     

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.     

Cryopreservation of goat oocytes and in vivo derived 2- to 4-cell embryos using the cryoloop (CLV) and solid-surface vitrification (SSV) methods

This study evaluated the efficiency and toxicity of two cryopreservation methods, solid-surface vitrification (SSV) and cryoloop vitrification (CLV), on in vitro matured oocytes and in vivo derived early stage goat embryos. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4% trehalose. Microdrops containing the oocytes were cryopreserved by dropping them on a cold metal surface that was partially immersed in liquid nitrogen. In the cryoloop method, oocytes were transferred onto a film of the CLV solution (20% DMSO, 20% EG, 10mg/ml Ficoll and 0.65 M sucrose) suspended in the cryoloop. The cryoloop was then plunged into the liquid nitrogen. In vivo derived embryos were vitrified using the same procedures. The SSV microdrops were warmed in a solution of 0.3M trehalose and those vitrified with CLV were warmed with incubation in 0.25 and 0.125 M sucrose. Oocytes and embryos vitrified by the SSV method had a significantly lower survival rate than the control (60 and 39% versus 100%, respectively; P<0.05), while the survival rate of CLV oocytes and embryos (89 and 88%, respectively) did not differ from controls. Cleavage and blastocyst rates of the surviving vitrified oocytes (parthenogenetically activated) and embryos (cultured for 9 days) were not significantly different (P>0.05) from the control nor did they differ between vitrification methods. Embryos vitrified with the CLV method gave rise to blastocysts (2/15). Our data demonstrated that the two vitrification methods employed resulted in acceptable levels of survival and cleavage of goat oocytes and embryos.     

Preimplantation development and viability of in vitro cultured rabbit embryos derived from in vivo fertilized gene-microinjected eggs: apoptosis and ultrastructure analyses

Microinjection (Mi) of gene constructs into pronuclei of fertilized eggs is a widely used method to generate transgenic animals. However, the efficiency of gene integration and expression is very low because of the low viability of reconstructed embryos resulting from cell fragmentation and cleavage arrest. As a consequence, only a few viable embryos integrate and express transgene. Since cellular fragmentation and cleavage stage arrest in embryos may be associated with apoptosis, we aimed to test the hypothesis that the low viability of Mi-derived eggs is caused by a high rate of apoptosis in embryos, as a result of the detrimental effect of Mi. Pronuclear stage eggs (19-20 hours post-coitum, hpc) were microinjected with several picolitres of DNA construct into the male pronucleus (gene-Mi); the intact eggs (non-Mi) or eggs microinjected with phosphate-buffered saline (PBS-Mi) served as controls. Epidermal growth factor (EGF; 0, 20 and 200 ng/ml) was added to the culture medium and the embryos were cultured up to 94-96 hpc. Apoptosis was detected using the TUNEL assay, and the ultrastructure was analysed using electron microscopy of Durcupan ACM thin sections of the embryo. Gene-Mi embryos had significantly lower (p < 0.05) blastocyst yields and a higher percentage of cleavage-arrested embryos than those in the non-Mi group. In gene-Mi groups, approximately 40% of all cleavage-stage-arrested embryos had fragmented blastomeres. Both gene-Mi- and PBS-Mi-derived blastocysts had a significantly higher TUNEL index (p < 0.001) and lower total cell number (p < 0.05) than the non-Mi embryos. Comparison of the quality of gene-Mi embryos with that of PBS-Mi embryos indicated that the deleterious effect of Mi on the embryo was caused by the Mi procedure itself, rather than DNA. EGF (at 20 ng/ml) had beneficial effects on the quality of gene-Mi-derived embryos, eliminating the influence of the Mi procedure on apoptosis and embryo cell number. Ultrastructural analysis confirmed a higher occurrence of apoptotic signs (nuclear membrane blebbing, areas with electron-dense material, numerous apoptotic bodies) in Mi-derived cleavage-arrested embryos compared with untreated or Mi-derived normal-looking embryos. These findings suggest an association between embryo cleavage arrest and apoptosis in Mi-derived embryos. Inclusion of EGF in the embryo culture medium can eliminate the detrimental effect of Mi on embryo quality.     

Development of 4-cell mouse embryos after re-vitrification

This paper reports studies on the effects of re-vitrification by the CPS (Closed Pulled Straw) method on the development of 4-cell stage mouse embryos. The procedure involved culturing 2-cell mouse embryos in G-1 medium until the 4-cell stage followed by the division of the normal 4-cell stage embryos into a control group (non-vitrified) and two experimental subgroups (vitrified and re-vitrified). Embryos in the vitrified subgroup were cryopreserved by the CPS vitrification method. In the second experimental subgroup (re-vitrified), embryos that were already vitrified were warmed and cryopreserved again by the same method. There was no significant reduction in the rate of blastocyst formation after vitrification and re-vitrification. However, re-vitrification reduced the total cell number, ICM (inner cell mass) percent and blastocyst diameter (P<0.05). These results showed that vitrification and re-vitrification by the CPS method did not negatively affect the development of vitrified-warmed 4-cell mouse embryos, whereas re-vitrification significantly reduced both the cell number and diameter of blastocysts.     

Double vitrification of rat embryos at different developmental stages using an identical protocol

The aim of the present investigation was to test the effectiveness of a method of vitrifying rat embryos at different stages of development (from early morula to expanding blastocyst) in a double vitrification procedure. Wistar rat embryos were vitrified and warmed in super-fine open-pulled straws (SOPS). Before being plunged into liquid nitrogen, the embryos were exposed to 40% ethylene glycol+0.75 M sucrose in TCM-199+20% fetal calf serum (FCS) for 20s at 38 degrees C. Subsequent warming and direct rehydration of the embryos was conducted in culture medium (TCM-199+20% FCS) at 38 degrees C. Early morula stage (7-10 blastomeres) embryos (n=358) were vitrified, warmed and cultured in vitro (EM group). Batches of these embryos were then cryopreserved again (revitrified) at the early blastocyst (EB group, n=87), blastocyst (B group, n=93) or expanding blastocyst stage (ExpB group, n=73). After the first (EM group) and repeated (EB, B, and ExpB groups) vitrification procedures, developmental rates of 81, 83, 34 and 76%, respectively were achieved (for EM-EB-ExpB P>0.1; for EM, EB, ExpB-B P<0.005). Our data demonstrate the possibility of using the described identical protocol for the SOPS vitrification of rat early morulae, early blastocysts and expanding blastocysts. The low survival rate of blastocysts subjected to double vitrification requires further investigation.     

Vitrification of isolated mice blastomeres using a closed loading device

Isolated blastomeres obtained by embryo biopsy serve mainly for preimplantation genetic screening. Blastomeres are undifferentiated embryonic cells that include all the embryo genetic information. A lot of developing technologies may benefit by the efficient cryopreservation of blastomeres for future potential use, especially for stem cell culture and differentiation control. We are hereby reporting for the first time the feasibility of preserving individual isolated blastomeres in microvolumes in a closed vitrification system. Using a cryotip and propagation in microvolumes, isolated mice blastomeres were vitrified and warmed with 100% post-warming survival.     

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirmed an individual effect of the bull as well as development rates of embryos produced with sorted sperm lower than embryos with unsorted sperm, independent of the culture system used. The cryoresistance to vitrification of embryos produced with sexed sperm did not differ from that of conventionally produced embryos (re-expansion rates at 24 and 48 h: 74.6% vs. 75.5%, and 64.5% vs. 68.1% for embryos produced with conventional and sorted sperm, respectively; hatching rates at 48 h: 63.55% vs. 55.5% for embryos produced with conventional and sorted sperm, respectively). Finally, no significant differences were found in pregnancy rates after the embryo transfer of fresh and vitrified/warmed blastocysts (52.8% vs. 42.0%, respectively; P > 0.05). Male and female embryos produced with sorted sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. Our culture system, coupled with the vitrification in fiber plugs, provides good quality sex-known embryos which survive vitrification at similar rates than embryos produced with conventional unsorted sperm; also it produces good pregnancy rates after transfer of sexed embryos both fresh and after vitrification and warming.     

A simple vitrification technique for sheep and goat embryo cryopreservation

The aim of this study was to evaluate pregnancy and embryo survival rate of vitrified in vivo produced Merino sheep and Criolla goat (morulae and blastocysts) embryos, using the plastic tips of micropipettes, as containers (Cryo-tips). The embryos were exposed, at room temperature, to two successive equilibration solutions for a period of 5 min and then to a vitrification solution (VS) for 30 s. Then embryos were then loaded in 1 μl VS, into a plastic micropipette tip, and plunged into liquid nitrogen. On thawing, the embryos were warmed (37 °C) and placed into cryoprotectant dilutions (three-step-process). In the ovine, the morula and blastocyst pregnancy rates (47.1% vs 50%) and embryo survival rates (41.2% vs 50%) recorded were similar for both embryonic stages. Unlike the sheep, no pregnancies were recorded in goat vitrified/thawed morulae embryos, following transfer. However, in contrast, goats receiving blastocysts recorded high rates of pregnancy and embryo survival (64% and 64%, respectively). This technique allows for easy handling of cryopreserved embryos, is simple and efficient in both ovine embryo stages and also for goat vitrified blastocysts. The technique has definite potential application.     

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.     

Vitrification of in vitro produced goat blastocysts: effects of oocyte donor age and development stage

This study examines the effectiveness of the cryotop vitrification method for the cryopreservation of goat blastocysts. To determine the effects of embryo development stage and donor age on in vitro survival rates, good-quality blastocysts from adult and prepubertal goats were sorted into non-expanded, expanded, hatching and completely hatched. In vitro produced (IVP) blastocysts were derived from prepubertal goat oocytes by slicing of ovaries from slaughtered animals while adult goat oocytes were collected by the laparoscopic ovum pick up (LOPU) method. Blastocysts were vitrified/warmed using the cryotop technique. Survival rates were determined in terms of blastocoele re-expansion at 3 and 20 h post-warming. For prepubertal goats, survival rates at 3 h post-warming were significantly higher when expanded blastocysts (78.3%) were vitrified/warmed compared to hatched blastocysts (57.4%), whereas non-expanded (62.5%) or hatching blastocysts (71.4%) showed similar rates. For adult goats, survival rates were significantly higher after warming in expanded (36.4%), hatching (75%) or hatched (50%) blastocysts when compared to non-expanded (0%) blastocysts. When survival rates were assessed at 20 h post-warming, no differences were observed when we compared non-expanded (45.8%), expanded (56.5%), hatching (64.3%) and hatched (50.5%) blastocysts from prepubertal goats; and for blastocysts from adult goats, survival rates were only significantly lower for the non-expanded stage (0%) compared to the other stages. For adult versus prepubertal blastocysts at the same developmental stage, our data indicate significantly higher survival rates at 3 h post-warming for non-expanded and expanded blastocysts from prepubertal goats over their counterparts from adult goats. At 20 h post warming, survival rates were only higher for non-expanded blastocysts from prepubertal goats versus adult goats. Collectively, our data reveal that blastocysts produced in vitro from prepubertal goats return similar survival rates regardless of their development stage, whereas blastocysts derived from adult goats are best for vitrification at the expanded, hatching or hatched stage.     

Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures

In this study, three different vitrification systems (open pulled straw: OPS; superfine open pulled straw: SOPS; and Vit-Master technology using SOPS: Vit-Master-SOPS) were compared in order to investigate the influence of cooling rate on in vitro development of vitrified/warmed porcine morulae, early blastocysts, or expanded blastocysts. Embryos were obtained surgically on Day 6 of the estrous cycle (D0 = onset of estrus) from weaned crossbred sows, classified and pooled according their developmental stage. A subset of embryos from each developmental stage was cultured to evaluate the in vitro development of fresh embryos; the remaining embryos were randomly allocated to each vitrification system. After vitrification and warming, embryos were cultured in vitro for 96 h in TCM199 with 10% fetal calf serum at 39 degrees C, in 5% CO(2) in humidified air. During the culture period, embryos were morphologically evaluated for their developmental progression. The developmental stage of embryos at collection affected the survival and hatching rates of vitrified/warmed embryos (P < 0.001). The vitrification system or the interaction of vitrification system and developmental stage had no effect on these parameters (P > 0.05). Vitrified expanded blastocysts showed the best development in vitro (P < 0.001), with survival and hatching rates similar to those of fresh expanded blastocysts. The hatching rate of fresh morula or early blastocyst stage embryos was higher than their vitrified counterparts. In conclusion, under our experimental conditions, cooling rates greater than 20,000 degrees C/min, as occurs when SOPS or Vit-Master-SOPS systems are used, do not enhance the efficiency of in vitro development of vitrified porcine embryos.